Methods, mixtures, kits and compositions pertaining to analyte determination

ABSTRACT

This invention pertains to methods, mixtures, kits and compositions pertaining to analyte determination by mass spectrometry using labeling reagents that comprise a nucleophilic reactive group that reacts with a functional group of an analyte to produce a labeled analyte. The labeling reagents can be used as isobaric sets, mass differential labeling sets or in a combination of isobaric and mass differential labeling sets.

PRIORITY AND RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/770,212, filed Jun. 28, 2007, now U.S. Pat. No. 7,906,341, whichclaims the benefit of U.S. Provisional Patent Application No.60/817,513, filed Jun. 30, 2006, each of which is incorporated byreference herein in its entirety.

The section headings used herein are for organizational purposes onlyand should not be construed as limiting the subject matter described inany way.

FIELD

This invention pertains to methods, mixtures, kits and compositionspertaining to analyte determination by mass spectrometry.

INTRODUCTION

This invention pertains to the determination of an analyte or analytesby mass analysis. An analyte can be any molecule of interest that can bereacted with an amine or hydrazine group to thereby form a stableadduct. For example, the reactive group of the analyte can be acarboxylic acid, aldehyde or ketone group wherein the adduct formed fromthe aldehyde or ketone group can be reduced to form the stable labeledanalyte. Non-limiting examples of analytes include, but are not limitedto, biologically important carboxylic acid compounds (e.g.,prostaglandins, fatty acids, carnitines, etc.), proteins, peptides,carbohydrates, lipids, amino acids, steroids and other small moleculeshaving a mass of less than 1500 daltons that comprise an appropriatereactive functional group. Analytes can be determined using uniquelabeling reagents that permit the relative and/or absolutequantification of the analytes in complex mixtures. The labelingreagents can be used in sets for the analysis of complex sample mixtureswherein the labeling reagents can be isobaric (including isomericisobars) and/or comprise labeling reagents of unique gross mass (i.e. bemass differential labeling reagents also known as ‘mass differentialtags’, herein).

For example, multiplexed qualitative and quantitative analysis ofbiologically important compounds (for example, carboxy acid compoundssuch as prostaglandins, fatty acids, carnitines, etc.), can be performedutilizing isobaric and/or mass differential tags, and, in someembodiments, multiple reaction monitoring (MRM) on triple quadrupole,linear ion trap instruments.

With reference to FIG. 1 a, labeling reagents of an isobaric set cancomprise a reporter moiety, a balance (or linker) moiety and a reactivegroup wherein the reactive group is substituted by the analyte in theanalyte reacted form of the composition. Examples of labeling reagentsand labeled analytes of this general formula have been disclosed in, forexample, published copending and commonly owned United States patentapplication Serial Nos. US 2004-0219685 A1, US 2005-0114042 A1, US2005-0147982 A1, US 2005-0147985 A1, US 2005-0147987 A1, US 2005-0148771A1, US 2005-0148773 A1 and US 2005-0148774 A1.

In some embodiments, isobaric (including isomeric isobars) labelingreagents can be used to label, for example, the analytes of two or moredifferent samples wherein the different labeling reagents of a set allhave the same gross mass but wherein each reporter moiety can beuniquely isotopically encoded such that each reporter moiety of the sethas a unique gross mass. Because all the reagents of the set can havethe same gross mass but can comprise a reporter moiety of unique grossmass, the balance (or linker) can generally (but not necessarily) alsocomprise one or more heavy atom isotopes to thereby “balance” the massof each unique reporter such that the reporter/linker combination ofeach labeling reagent of the set has the same gross mass.

In some embodiments, mass differential labeling reagents (i.e. massdifferential tags) can be used to label, for example, the analytes oftwo or more different samples wherein the different labeling reagents ofa set all have a distinct mass (i.e. can all possess a known massdifference as compared with other reagents of the set). Because all thereagents of the set can have a known mass difference, the labelingreagents need not be fragmented in order to quantify the relativeamounts of like analytes in two different samples. However, in someembodiments, the labeling reagents can be fragmentable.

As discussed in more detail below, compounds of various general formulas(including sets comprising compounds of the same general structuralformula but for their isotopic coding) can be prepared in both isobaricand/or mass differential sets. Generally, sets can be used either as anisobaric set of labels or as a mass differential set of labels althoughthe simultaneous use of a set of isobaric labeling reagents incombination with a set of mass differential labeling reagents in thesame experiment is envisioned as an embodiment of this invention.

An example of a new labeling reagent (or labeled analyte), as discussedmore thoroughly herein, is illustrated in FIG. 1 b. Although illustratedin unsubstituted form (except for R₁ and R₂), it is to be understoodthat the labeling reagent can be substituted or unsubstituted. In theillustration, certain bonds are shown as being fragmented to therebyrelease at least the unique reporter moiety, and optionally the balancemoiety, from the labeling reagent or labeled analyte. For the labeledanalytes that have been labeled with isobaric labeling reagents, eachunique reporter ion (sometimes referred to as the signature ion)observed in the mass spectrometer (typically in MS^(n) analysis whereinn is an integer greater than 1) can be used to quantify the amount ofanalyte in a sample or sample mixture. For labeled analytes that havebeen labeled with mass differential labeling reagents, quantificationcan be determined by the relative intensity of the labeled analytes inMS¹ analysis.

FIG. 4 a illustrates two sets of 4 different encoded versions oflabeling reagents with the set of AI to AIV having the basic structureillustrated in FIG. 1 b (for example R₁ and R₂ can be, independently ofthe other, hydrogen or methyl), wherein the asterisk (*) is used toindicate where a ¹³C atom is substituted for a ¹²C atom, where a ¹⁵Natom is substituted for a ¹⁴N atom or where an ¹⁸O is substituted for a¹⁶O atom, as appropriate. These sets can be used to facilitate at leasta 4-plex experiment. However, with further substitution of the compoundswith heavy atom isotopes, it is expected that more than 4 differentcompounds can be prepared such that greater than a 4-plex experimentcould be performed.

Generally, labeling reagents, labeled analytes and some intermediates tothe labeling reagents and/or labeled analytes can be represented bycompounds of formula I;

including a salt form thereof and/or a hydrate form thereof, wherein Zcan be hydrogen or a covalently linked analyte and wherein the atoms orgroups X₁, R₁, R₂, Y, J, and K are described in more detail below.

Accordingly, in some embodiments analytes can be labeled by reaction ofthe analyte with a labeling reagent represented by compounds of formulaI′;

including a salt form thereof and/or a hydrate form thereof, wherein theatoms or groups R₁, R₂, Y, J, and K are described in more detail below.In some embodiments, the labeling reagents can be used in sets, whereinthe sets comprise isomeric and/or isobaric compounds, whereby thelabeled analytes can likewise be isomeric and/or isobaric. In someembodiments, the labeling reagents can be used in set, wherein the setscomprise mass differential labeling reagents (i.e. labeling reagents ofdifferent gross mass). In some embodiments, isobaric (including isobaricisomers) and mass differential labeling reagents are used together.

Further, in some embodiments a labeled analyte therefore can berepresented by formula I″;

including a salt form thereof and/or a hydrate form thereof, wherein Z″represents the analyte covalently linked to the labeling reagent andwherein the atoms or groups R₁, R₂, Y, J, and K are described in moredetail below.

As described herein, sets of two, three, four, or more, isobaric and/ormass differential labeling reagents can be made thereby permittingexperiments of 4-plex or greater. For example, it is possible tosimultaneously identify and/or quantify an analyte in 4 (or more)different samples that have each been differentially labeled and thenmixed. For an isobaric set of labels, quantification can be achieved bydetermination of the relative abundance of each unique reporter ionassociated with each different labeling reagent of the isobaric set. Fora mass differential set, quantification can be achieved by determinationof the relative abundance of each labeled analyte associated with eachdifferent labeling reagent of the mass differential set.

Thus, embodiments of this invention are particularly well suited for themultiplex analysis of complex sample mixtures. For example, someembodiments of this invention can be used in proteomic analysis and/orgenomic analysis as well as for correlation studies related to genomicand/or proteomic analysis. Some embodiments of this invention can alsobe used for small molecule analysis, such as for carnitine,carbohydrate, lipid, steroid, vitamin, prostaglandin, fatty acid, and/oramino acid analysis. Experimental analysis for which the isobaric and/ormass differential reagents can be used includes, but is not limited to,time course studies, biomarker analysis, multiplex proteomic analysis,mudpit experiments, affinity pull-downs, determination ofpost-translational modifications (PTMs) (see for example publishedUnited States Patent Application No. US 2005-0208550 A1) and multiplecontrol experiments.

DRAWINGS

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way.

FIG. 1 a is an illustration of the elements of a labeling reagent orlabeled analyte and some fragmentation characteristics.

FIG. 1 b is an illustration of the general elements of an exemplaryN-methyl piperazine based labeling reagent or labeled analyte and somefragmentation characteristics.

FIG. 1 c is an illustration of the general elements of a labeled analyteof the identified general formula and some fragmentationcharacteristics.

FIG. 2 is an illustration of the general structure of two differentlabeling reagents (i.e. structure A and structure B).

FIG. 3 is an illustration of possible fragmentation properties ofanalytes labeled with labeling reagents according to structures A and B(represented as labeled analytes A″ and B″) as well as an illustrationof the mass of possible fragments generated therefrom.

FIG. 4 a is an illustration of two possible sets of isobaric labelingreagents (based upon structure A and structure B, respectively) each ofwhich produces the same reporter ion set.

FIG. 4 b is an illustration of possible structures for the reporter ions114-117 mentioned in FIG. 4 a as well as the possible structures for thenon-encoded (i.e. “cold”) reporter ion 113.

FIGS. 5 a and 5 b are illustrations of two possible sets of massdifferential labeling reagents (based upon structure A and structure B,respectively)

FIGS. 6 a and 6 b are illustrations of the processes of labelinganalytes with the exemplary compounds of structures A and B,respectively.

All literature and similar materials cited in this application,including but not limited to, patents, patent applications, articles,books, treatises, and internet web pages, regardless of the format ofsuch literature and similar materials, are expressly incorporated byreference herein in their entirety for any and all purposes.

DEFINITIONS

For the purposes of interpreting this specification, the followingdefinitions will apply and whenever appropriate, terms used in thesingular will also include the plural and vice versa. In the event thatany definition set forth below conflicts with the usage of that word inany other document, including any document incorporated herein byreference, the definition set forth below shall always control forpurposes of interpreting this specification and its associated claimsunless a contrary meaning is clearly intended (for example ininterpreting the document where the term is originally used). The use of“or” herein means “and/or” unless stated otherwise or where the use of“and/or” is clearly inappropriate. The use of “a” herein means “one ormore” unless stated otherwise or where the use of “one or more” isclearly inappropriate. The use of “comprise,” “comprises,” “comprising”“include,” “includes,” and “including” are interchangeable and notintended to be limiting. Furthermore, where the description of one ormore embodiments uses the term “comprising,” those skilled in the artwould understand that in some specific instances, the embodiment orembodiments can be alternatively described using language “consistingessentially of” and/or “consisting of.” It should also be understoodthat in some embodiments the order of steps or order for performingcertain actions is immaterial so long as the present teachings remainoperable. Moreover, in some embodiments two or more steps or actions canbe conducted simultaneously.

a.) As used herein, “analyte” refers to a molecule of interest that maybe determined. Non-limiting examples of analytes include, but are notlimited to, proteins, peptides, (either DNA or RNA), carbohydrates,lipids, amino acids, steroids, vitamins, prostaglandins, fatty acids,carnitines and other small molecules with a molecular weight of lessthan 1500 daltons (Da). The source of the analyte, or the samplecomprising the analyte, is not a limitation as it can come from anysource. The analyte or analytes can be natural or synthetic.Non-limiting examples of sources for the analyte, or the samplecomprising the analyte, include cells or tissues, or cultures (orsubcultures) thereof. Non-limiting examples of analyte sources include,but are not limited to, crude or processed cell lysates, body fluids,tissue extracts, cell extracts or fractions (or portions) from aseparations process such as a chromatographic separation, a 1Delectrophoretic separation, a 2D electrophoretic separation or acapillary electrophoretic separation. Body fluids include, but are notlimited to, blood, urine, feces, spinal fluid, cerebral fluid, amnioticfluid, lymph fluid or a fluid from a glandular secretion. By processedcell lysate we mean that the cell lysate is treated, in addition to thetreatments needed to lyse the cell, to thereby perform additionalprocessing of the collected material. For example, the sample can be acell lysate comprising one or more analytes that are peptides formed bytreatment of the cell lysate with one or more proteolytic enzymes tothereby digest precursor peptides and/or proteins.

b.) Except as when clearly not intended based upon the context in whichit is being used (e.g. when made in reference to a specific structurethat dictates otherwise), “ester” refers to both an ester and/or athioester.

c.) As used herein, “fragmentation” refers to the breaking of a covalentbond.

d.) As used herein, “fragment” refers to a product of fragmentation(noun) or the operation of causing fragmentation (verb).

e.) As used herein, “hydrate form” refers to any hydration state of acompound or a mixture or more than one hydration state of a compound.For example, a labeling reagent discussed herein can be a hemihydrate, amonohydrate, a dihydrate, etc. Moreover, a sample of a labeling reagentdescribed herein can comprise monohydrate, dihydrate and hemihydrateforms.

f.) As used herein, a halogen group refers to —F, —CI, —Br, or —I.

g.) As used herein with respect to a compound, “isotopically enriched”refers to a compound (e.g. labeling reagent) that has been enriched withone or more heavy atom isotopes (e.g. stable isotopes such as deuterium(‘D’), ¹³C, ¹⁵N, ¹⁸O, ³⁷Cl or ⁸¹Br). In some embodiments, unstableisotopes can also be used (e.g. ¹⁴C or ³H). By “enriched” we mean thatthe amount of heavy atom isotope exceeds natural isotopic abundance. Invarious embodiments, the isotopically enriched compound can comprise 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30 or more isotopically enriched sites.

Because isotopic enrichment is not 100% effective, there can beimpurities in a sample of the compound that are of lesser states ofenrichment and these will have a lower mass. Likewise, because ofover-enrichment (e.g. undesired enrichment) and because of naturalisotopic abundance, there can be impurities in a sample of the compoundthat are of greater mass. In some embodiments, each incorporated heavyatom isotope can be present at an isotopically enriched site in at least80 percent isotopic purity. In some embodiments, each incorporated heavyatom isotope can be present at an isotopically enriched sited in atleast 93 percent isotopic purity. In some embodiments, each incorporatedheavy atom isotope can be present at an isotopically enriched site in atleast 96 percent isotopic purity. In some embodiments, each incorporatedheavy atom isotope can be present at an isotopically enriched site in atleast 98 percent purity.

h.) As used herein, “isotopically enriched site” refers to the positionin a compound where a heavy atom isotope is substituted for a lightversion of the atom (e.g. substitution of ¹³C for ¹²C, ¹⁸O for ¹⁶O, ¹⁵Nfor ¹⁴N or ¹⁴N or deuterium for hydrogen).

i.) As used herein with respect to a compound, “light” refers to thecompound as not being enriched with a heavy atom isotope. As used hereinwith respect to an atom, “light” refers to the lowest mass isotope ofthe atom. As used herein with respect to a compound, “heavy” refers tothe compound as being enriched with at least one heavy atom isotope. Asused herein with respect to an atom, “heavy” refers to a heavy massisotope of the atom.

j.) As used herein, “labeling reagent” refers to a moiety suitable tomark an analyte for determination. The term label is synonymous with theterms tag and mark and other equivalent terms and phrases. For example,a labeled analyte can also be referred to as a tagged analyte or amarked analyte. Accordingly the terms “label”, “tag”, “mass tag”, “mark”and derivatives of these terms, are equivalent and interchangeable andrefer to a moiety suitable to mark, or that has marked, an analyte fordetermination. Sometimes a labeling reagent can be referred to a taggingreagent, a mass tagging reagent or simply as a mass tag (e.g. massdifferential tag).

k.) As used herein, “natural isotopic abundance” refers to the level (ordistribution) of one or more heavy isotopes found in a compound basedupon the natural prevalence of an isotope or isotopes in nature. Forexample, a natural compound obtained from living plant matter cantypically contain about 1.08% ¹³C relative to ¹²C.

l.) As used herein, isobars are structurally and chemicallyindistinguishable compounds (except for isotopic content and/ordistribution of heavy atom isotopes) of the same nominal gross mass. By“chemically indistinguishable” we mean that the isobars comprise thesame general chemical structure (but for the distribution of heavy atomisotopes) and possess substantially the same chemical reactivity andseparations properties.

m.) As used herein, “support”, “solid support”, “solid carrier” or“resin” means any solid phase material. Solid support encompasses termssuch as “support”, “synthesis support”, “solid phase”, “surface”“membrane” and/or “support”. A solid support can be composed of organicpolymers such as polystyrene, polyethylene, polypropylene,polyfluoroethylene, polyethyleneoxy, and polyacrylamide, as well asco-polymers and grafts thereof. A solid support can also be inorganic,such as glass, silica, controlled-pore-glass (CPG), or reverse-phasesilica. The configuration of a solid support can be in the form ofbeads, spheres, particles, granules, a gel, a membrane or a surface.Surfaces can be planar, substantially planar, or non-planar. Solidsupports can be porous or non-porous, and can have swelling ornon-swelling characteristics. A solid support can be configured in theform of a well, depression or other container, vessel, feature orlocation. A plurality of solid supports can be configured in an array atvarious locations, addressable for robotic delivery of reagents, or bydetection methods and/or instruments.

n.) As used herein, “sample, or a fraction thereof” or “sample fraction”can be used to refer to a fraction of a sample. The fraction of thesample can be generated either by simply withdrawing a fraction of asample else it can be generated by performing a separations process thatresults in the sample being fractionated into two or more fractions.Unless the content of the description indicates otherwise, these phrasesare equivalent and interchangeable and refer to any type of creation ofa fraction (or portion) of a sample.

o.) As used herein, “signature ion” and “reporter ion” areinterchangeable and both refer to the reporter ion of unique massproduced from the reporter moiety by fragmentation of a labeling reagentor labeled analyte. The signature ion or reporter ion can be used toidentify the unique labeling reagent used to label an analyte and itspeak intensity in MS/MS analysis (or MS^(n) analysis) can be correlatedwith the amount of labeled analyte present in the sample that isanalyzed. As used herein, the signature ion or reporter ion is sometimesmerely referred to as a reporter. As used herein, the reporter moiety isalso sometimes merely referred to a reporter. It is to be understoodthat the reporter moiety refers to the group attached to a labelingreagent, labeled analyte or fragment thereof and the reporter ion refersto the fragment ion generated upon fragmentation of the bond that linksthe reporter moiety to the labeling reagent, labeled analyte or afragment thereof. Accordingly, the context in which the word “reporter”is used can indicate its intended meaning. It also is to be understoodthat the phrase “unique reporter moiety” is equivalent to, andinterchangeable with, “reporter moiety of unique mass” and that “uniquereporter ion” is equivalent to, and interchangeable with, “reporter ionof unique mass”.

p.) As used herein, the term “salt form” includes a salt of a compoundor a mixture of salts of a compound. In addition, zwitterionic forms ofa compound are also included in the term “salt form.” Salts of compoundshaving an amine, or other basic group can be obtained, for example, byreaction with a suitable organic or inorganic acid, such as hydrogenchloride, hydrogen bromide, acetic acid, perchloric acid and the like.Compounds with a quaternary ammonium group may also contain acounteranion such as chloride, bromide, iodide, acetate, perchlorate andthe like. Salts of compounds having a carboxylic acid, or other acidicfunctional group, can be prepared by reacting the compound with asuitable base, for example, a hydroxide base. Accordingly, salts ofacidic functional groups may have a countercation, such as sodium,potassium, magnesium, calcium, etc.

q.) As used herein, “synthetic compound” refers to a compound that iscreated by manipulation of processes including the manipulation ofnaturally occurring pathways. Thus, a synthetic compound can be producedusing synthetic chemistry techniques. However, as used herein,“synthetic compound” is also intended to include compounds that areproduced, for example, by enzymatic methods, including for example,feeding isotopically enriched compounds to organisms, such as bacteriaor yeast, that alter them to thereby produce the isotopically enrichedlabeling reagents, or intermediates of the labeling reagents, describedherein.

r.) As used herein, “synthetically enriched” or “enriched synthetically”refers to the manipulation of a synthetic or natural process to therebyproduce a synthetic compound such as the isotopically enriched labelingreagents, or intermediates to the labeling reagents, described herein.

s.) It is well accepted that the mass of an atom or molecule can beapproximated, often to the nearest whole number atomic mass unit or thenearest tenth or hundredth of an atomic mass unit. As used herein,“gross mass” refers to the absolute mass as well as to the approximatemass within a range where the use of isotopes of different atom typesare so close in mass that they are the functional equivalent for thepurpose of balancing the mass of the reporter and/or linker moieties (sothat the gross mass of the reporter/linker combination is the samewithin a set or kit of isomeric and/or isobaric labeling reagents)whether or not the very small difference in mass of the differentisotopes types used can be detected.

For example, the common isotopes of oxygen have a gross mass of 16.0(actual mass 15.9949) and 18.0 (actual mass 17.9992), the commonisotopes of carbon have a gross mass of 12.0 (actual mass 12.00000) and13.0 (actual mass 13.00336) and the common isotopes of nitrogen have agross mass of 14.0 (actual mass 14.0031) and 15.0 (actual mass 15.0001).Whilst these values are approximate, one of skill in the art canappreciate that if one uses the ¹⁸O isotope at an isotopically enrichedsite within one label of a set, the additional 2 mass units (over theisotope of oxygen having a gross mass of 16.0) can, for example, becompensated for in a different label of the set comprising ¹⁶O byincorporating, elsewhere in the label, two carbon ¹³C atoms, instead oftwo ¹²C atoms, two ¹⁵N atoms, instead of two ¹⁴N atoms or even one ¹³Catom and one ¹⁵N atom, instead of a ¹²C and a ¹⁴N, to compensate for the¹⁸O. In this way two different labels of an isobaric set can have thesame gross mass since the very small actual differences in mass betweenthe use of two ¹³C atoms (instead of two ¹²C atoms), two ¹⁵N atoms(instead of two ¹⁴N atoms), one ¹³C and one ¹⁵N (instead of a ¹²C and¹⁴N) or one ¹⁸O atom (instead of one ¹⁶O atom), to thereby achieve anincrease in mass of two Daltons in all of the labels of the set or kit,is not an impediment to the nature of the analysis.

It is clear that the distribution of the same heavy atom isotopes withina structure is not the only consideration for the creation of sets ofisobaric and/or mass differential labeling reagents. It is possible tomix heavy atom isotope types to achieve isobars or mass differentiallabels of a desired gross mass. In this way, both the selection(combination) of heavy atom isotopes as well as their distribution isavailable for consideration in the production of the isobaric and/ormass differential labeling reagents useful for embodiments of thisinvention.

t.) As used herein, the term “alkyl” refers to a straight chained orbranched C₂-C₈ hydrocarbon or a cyclic C₃-C₈ hydrocarbon (i.e. acycloalkyl group such as a cyclopropyl group, a cyclobutyl group, acyclopentyl group, a cyclohexyl group or a cyclohexylmethylene group)that can be completely saturated. When used herein, the term “alkyl”refers to a group that may be substituted or unsubstituted. In someembodiments, the term “alkyl” is also intended to refer to thosecompounds wherein one or more methylene groups in the alkyl chain can bereplaced by a heteroatom such as —O—, —Si— or —S—. In some embodiments,alkyl groups can be straight chained or branched C₂-C₆ hydrocarbons orcyclic C₃-C₆ hydrocarbons that can be completely saturated.

u.) As used herein, the term “alkylene” refers to a straight or branchedalkyl chain or a cyclic alkyl group that comprises at least two pointsof attachment to at least two moieties

(e.g., —{CH₂}— (methylene), —{CH₂CH₂}—, (ethylene), etc., wherein thebrackets indicate the points of attachment. When used herein the term“alkylene” refers to a group that may be substituted or unsubstituted.In some embodiments, the term “alkylene” is also intended to refer tothose compounds wherein one or more methylene groups, if any, in analkyl chain of the alkylene group can be replaced by a heteroatom suchas —O—, —Si— or —S—. In some embodiments, an alkylene group can be aC₁-C₁₀ hydrocarbon. In some embodiments, an alkylene group can be aC₂-C₆ hydrocarbon.

v.) As used herein, the term “alkenyl” refers to a straight chained orbranched C₂-C₈ hydrocarbon or a cyclic C₃-C₈ hydrocarbon that comprisesone or more double bonds. When used herein, the term “alkenyl” refers toa group that can be substituted or unsubstituted. In some embodiments,the term “alkenyl” is also intended to refer to those compounds whereinone or more methylene groups, if any, in an alkyl chain of the alkenylgroup can be replaced by a heteroatom such as —O—, —Si— or —S—. In someembodiments, alkenyl groups can be straight chained or branched C₂-C₆hydrocarbons or cyclic C₃-C₆ hydrocarbons that comprise one or moredouble bonds.

w.) As used herein, the term “alkenylene” refers to an alkenyl groupthat comprises two points of attachment to at least two moieties. Whenused herein the term “alkenylene” refers to a group that may besubstituted or unsubstituted. In some embodiments, the term “alkenylene”is also intended to refer to those compounds wherein one or moremethylene groups, if any, in an alkyl chain of the alkenylene group canbe replaced by a heteroatom such as —O—, —Si— or —S—.

x.) As used herein, the term “alkynyl” refers to a straight chained orbranched C₂-C₈ hydrocarbon or a cyclic C₃-C₃ hydrocarbon that comprisesone or more triple bonds. When used herein, the term “alkynyl” refers toa group that may be substituted or unsubstituted. In some embodiments,the term “alkynyl” is also intended to refer to those compounds whereinone or more methylene groups, if any, in an alkyl chain of the alkynylgroup can be replaced by a heteroatom such as —O—, —Si— or —S—. In someembodiments, alkynyl groups can be straight chained or branched C₂-C₆hydrocarbons or cyclic C₃-C₆ hydrocarbons that have one or more triplebonds.

y.) As used herein, the term “alkynylene” refers to an alkynyl groupthat comprises two points of attachment to at least two moieties. Whenused herein the term “alkynylene” refers to a group that may besubstituted or unsubstituted. In some embodiments, the term “alkynylene”is also intended to refer to those compounds wherein one or moremethylene groups, if any, in an alkyl chain of the alkynylene group canbe replaced by a heteroatom such as —O—, —Si— or —S—.

z.) As used herein, the term “aliphatic” refers to any of the straight,branched, or cyclic alkyl, alkenyl, and alkynyl moieties as definedabove. When used herein the term “aliphatic” refers to a group that maybe substituted or unsubstituted.

aa.) As used herein, the term “aryl”, either alone or as part of anothermoiety (e.g., arylalkyl, etc.), refers to carbocyclic aromatic groupssuch as phenyl. Aryl groups also include fused polycyclic aromatic ringsystems in which a carbocyclic aromatic ring is fused to anothercarbocyclic aromatic ring (e.g., 1-naphthyl, 2-naphthyl, 1-anthracyl,2-anthracyl, etc.) or in which a carbocylic aromatic ring is fused toone or more carbocyclic non-aromatic rings (e.g., tetrahydronaphthylene,indan, etc.). As used herein, the term “aryl” refers to a group that maybe substituted or unsubstituted.

ab.) As used herein, the term “heteroaryl,” refers to an aromaticheterocycle that comprises 1, 2, 3 or 4 heteroatoms selected,independently of the others, from nitrogen, sulfur and oxygen. As usedherein, the term “heteroaryl” refers to a group that may be substitutedor unsubstituted. A heteroaryl may be fused to one or two rings, such asa cycloalkyl, an aryl, or a heteroaryl ring. The point of attachment ofa heteroaryl to a molecule may be on the heteroaryl, cycloalkyl,heterocycloalkyl or aryl ring, and the heteroaryl group may be attachedthrough carbon or a heteroatom. Examples of heteroaryl groups includeimidazolyl, pyrrolyl, pyridinyl, pyrimidyl, pyrazinyl, pyridazinyl,quinolyl, isoquinolinyl, indazolyl, indolizinyl, imidazopyridinyl,pyrazolyl, triazolyl, tetrazolyl, indolyl, tetrahydroindolyl,azaindolyl, imidazopyridyl, quinazolinyl, purinyl, pyrrolo[2,3]pyrimidylor pyrazolo[3,4]pyrimidyl, each of which can be optionally substituted.

ac.) As used herein, the term “arylene” refers to an aryl or heteroarylgroup that comprises at least two points of attachment to at least twomoieties (e.g., phenylene, etc.). The point of attachment of an arylenefused to a carbocyclic, non-aromatic ring may be on either the aromatic,non-aromatic ring. As used herein, the term “arylene” refers to a groupthat may be substituted or unsubstituted.

ad.) As used herein, the term “arylalkyl” refers to an aryl orheteroaryl group that is attached to another moiety via an alkylenelinker. As used herein, the term “arylalkyl” refers to a group that maybe substituted or unsubstituted. In some embodiments, the term“arylalkyl” is also intended to refer to those compounds wherein one ormore methylene groups, if any, in the alkyl chain of the arylalkyl groupcan be replaced by a heteroatom such as —O—, —Si— or —S—.

ae.) As used herein, the term “arylalkylene” refers to an arylalkylgroup that has at least two points of attachment to at least twomoieties. The second point of attachment can be on either the aromaticring or the alkylene group. As used herein, the term “arylalkylene”refers to a group that may be substituted or unsubstituted. In someembodiments, the term “arylalkylene” is also intended to refer to thosecompounds wherein one or more methylene groups, if any, in the alkylchain of the arylalkylene group can be replaced by a heteroatom such as—O—, —Si— or —S—. When an arylalkylene is substituted, the substituentsmay be on either or both of the aromatic ring or the alkylene portion ofthe arylalkylene.

af.) As used herein, the terms “optionally substituted” and “substitutedor unsubstituted” are equivalent and interchangeable. Suitablesubstituents for any an alkyl, an alkylene, an alkenyl, an alkenylene,an alkynyl, an alkynylene, an aryl, an aryl alkyl, an arylene, aheteroaryl or an arylalkylene group includes any substituent that isstable under the reaction conditions used in embodiments of thisinvention. Non limiting examples of suitable substituents can include:an alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec butyl, t-butyl, cyclohexyl, etc.) group, a haloalkyl (e.g.,trifluoromethyl, 2,2,2-trifluoroethyl-, etc.) group, an alkoxy (e.g.,methoxy, ethoxy, etc.) group, an aryl (e.g., phenyl) group, an arylalkyl(e.g., benzyl) group, a nitro group, a cyano group, a quaternizednitrogen atom or a halogen group (e.g., fluorine, chlorine, bromineand/or iodine) group.

In addition, any portion of an alkyl, an alkylene, an alkenyl, analkenylene, an alkynyl, an alkynylene, an aryl, an aryl alkyl, anarylene, a heteroaryl or an arylalkylene group may also be substitutedwith ═O or ═S.

ag.) As used herein, the term “active ester” refers to compounds thatcan react readily under basic conditions with amines, alcohols andcertain thiols to provide amides, esters and thioesters, respectively.Additional reference is made to: Leo A Paquette, Encyclopedia ofReagents for Organic Synthesis, Vol. 2, John Wiley and Sons, New York,1995 as evidence that “active ester” is a term well-established in fieldof organic chemistry.

ah.) As used herein, the term “heterocyclic ring” refers to any cyclicmolecular structure comprising atoms of at least two different elementsin the ring or rings. Additional reference is made to: Oxford Dictionaryof Biochemistry and Molecular Biology, Oxford University Press, Oxford,1997 as evidence that “heterocyclic ring” is a term well-established infield of organic chemistry.

ai.) As used herein, the term “leaving group” refers to any atom orgroup, charged or uncharged, that departs during a substitution ordisplacement reaction from what is regarded as the residual or main partof the substrate of the reaction. Additional reference is made to:Oxford Dictionary of Biochemistry and Molecular Biology, OxfordUniversity Press, Oxford, 1997 as evidence that “leaving group” is aterm well-established in field of organic chemistry.

aj.) As used herein, the term “protecting group” refers to a chemicalgroup that is reacted with, and bound to, a functional group in amolecule to prevent the functional group from participating insubsequent reactions of the molecule but which group can subsequently beremoved to thereby regenerate the unprotected functional group.Additional reference is made to: Oxford Dictionary of Biochemistry andMolecular Biology, Oxford University Press, Oxford, 1997 as evidencethat “protecting group” is a term well-established in field of organicchemistry.

DESCRIPTION OF VARIOUS EMBODIMENTS

It is to be understood that the discussion set forth below in this“General” section can pertain to some, or to all, of the variousembodiments of the invention described herein.

I. General

Labeling Reagent(s):

A labeling reagent can comprises a reporter moiety, a balance moiety (orlinker moiety) and a reactive group (FIG. 1 a). Labeling reagents can bereacted with analytes to thereby produce labeled analytes. In someembodiments, the labeling reagents are organized into sets. In someembodiments, the sets can be isomeric and/or isobaric. In someembodiments the sets can be mass differential. In some embodiments, thesets can be both isobaric and/or isomeric and mass differential.

In some embodiments, the labeling reagents can have the generalstructure A or B as illustrated in FIG. 2. Labeling reagents of generalstructures A and B can be reacted with analytes to form structures A″and B″ as illustrated in FIG. 3. In some embodiments, the labeledanalytes can be fragmented in a mass spectrometer. Some possiblefragments and their respective masses can be found in FIG. 3. Twopossible sets of isobaric labeling reagents of general structure A and Bcan be found in FIG. 4 a, with the possible reporter ions illustrated inFIG. 4 b. Two sets of mass differential tags based upon generalstructures A and B can be found in FIGS. 5 a and 5 b. FIG. 6 a and FIG.6 b illustrate the labeling of an analyte using labeling reagents ofgeneral structure A and B.

The Reactive Group:

The reactive group (sometimes represented by use of the shorthand “RG”)of the labeling reagent or reagents used in the method, mixture, kitand/or composition embodiments can be a nucleophilic group that iscapable of reacting with one or more functional groups of one or morereactive analytes of a sample. It is to be understood that in someembodiments, the reactive group may be considered to include an atom orgroup associated with the linker (balance).

It is to be understood that when the reactive group is represented bysome of the specific moieties discussed below, the analyte may be linkedto the linker (balance) through one or more additional atoms or groupsthat may, or may not, be considered to be part of the linker (balance).

The reactive group can be preexisting or it can be prepared in-situ.In-situ preparation of the reactive group can proceed in the absence ofthe reactive analyte or it can proceed in the presence of the reactiveanalyte. In some embodiments, the reactive group can be generatedin-situ by the in-situ removal of a protecting group. Consequently, anyexisting or newly created reagent or reagents that can effect thederivatization of analytes by the reaction of nucleophiles and/orelectrophiles are contemplated by the method, mixture, kit and/orcomposition embodiments of this invention.

Where the reactive group of the labeling reagent is a nucleophile, itcan react with a suitable electrophilic group of the analyte oranalytes. Numerous pairs of suitable nucleophilic groups andelectrophilic groups are known and often used in the chemical andbiochemical arts. Non-limiting examples of reagents comprising suitablenucleophilic groups that can be coupled to analytes (e.g. such asbiologically important carboxy acid compounds (e.g., prostaglandins,fatty acids, carnitines, etc.), proteins, peptides, carbohydrates,lipids, steroids or other small molecules having a mass of less that1500 daltons) to effect their derivatization, are described in thePierce Life Science & Analytical Research Products Catalog & Handbook (aPerstorp Biotec Company), Rockford, Ill. 61105, USA. Other suitablereagents are well known in the art and are commercially available fromnumerous other vendors such as Sigma-Aldrich.

In some embodiments, the reactive group of the labeling reagent can be anucleophile such as an amine group or hydrazine group. In someembodiments, the nucleophilic reactive group can be an aminoalkyl groupor alkyl hydrazine group.

The Reporter Moiety:

The reporter moiety (sometimes represented by use of the shorthand “RP”)of the labeling reagent or reagents used in embodiments of thisinvention can be a group that has a unique mass (or mass to charge ratioin a mass spectrometer) that can be determined. Accordingly, in someembodiments, each reporter moiety of a set of isomeric and/or isobariclabeling reagents has a unique gross mass, and its correspondingsignature ion, is different for each labeling reagent of the set.

Different reporter moieties can comprise one or more heavy atom isotopesto achieve their unique gross mass. For example, isotopes of carbon(¹²C, ¹³C and ¹⁴C), nitrogen (¹⁴N and ¹⁵N), oxygen (¹⁶O and ¹⁸O), sulfur(³²S and ³⁴S) or hydrogen (hydrogen, deuterium and tritium) exist andcan be used in the preparation of a diverse group of reporter moieties.These are not limiting as other light and heavy atom isotopes can alsobe used in the reporter moieties. Basic starting materials suitable forpreparing reporter moieties comprising light and heavy atom isotopes areavailable from various commercial sources such as Cambridge IsotopeLaboratories, Andover, Mass. (See: list of “basic starting materials” atwww.isotope.com) and Isotec (a division of Sigma-Aldrich). CambridgeIsotope Laboratories and Isotec will also prepare desired compoundsunder custom synthesis contracts. Id.

The reporter moiety can either comprise a fixed charge or can becomeionized during the analysis process. Because the reporter moiety caneither comprises a fixed charge or can become ionized, the labelingreagent might be isolated or be used to label the reactive analyte in asalt (or a mixture of salts) or zwitterionic form. Ionization of thereporter moiety (or reporter ion) facilitates its determination in amass spectrometer. Accordingly, the presence of the reporter moiety in alabeled analyte can be determined as a fragment ion, sometimes referredto as a signature ion (or reporter ion).

When ionized, the signature ion (i.e. reporter ion) can comprise one ormore net positive or negative charges. Thus, the reporter ion cancomprise one or more acidic groups and/or basic groups since such groupscan be easily ionized in a mass spectrometer. For example, the reportermoiety can comprise one or more basic nitrogen atoms (positive charge)and/or one or more ionizable acidic groups such as a carboxylic acidgroup, sulfonic acid group or phosphoric acid group (negative charge)provided that on balance there are more of one or the other of the basicor acidic groups such that the reporter moiety produces a reporter ioncomprising a net positive or negative charge. Non-limiting examples ofreporter moieties comprising at least one basic nitrogen includesubstituted or unsubstituted morpholine, piperidine or piperazinecontaining compounds.

A unique reporter moiety can be associated with a sample of interestthereby labeling one or multiple analytes of that sample with saidunique reporter moiety. In this way information about the uniquereporter moiety (generally detected as a signature ion (i.e. reporterion) in a mass spectrometer) can be associated with information aboutone or all of the analytes of said sample.

However, the unique reporter moiety need not be physically linked to ananalyte when the signature ion is determined. Rather, the unique grossmass of the signature ion can, for example, be determined in a secondmass analysis of a tandem mass analyzer, after ions of the labeledanalyte are fragmented to thereby produce daughter fragment ions andsignature ions.

The determined signature ion(s) can be used to identify the sample fromwhich a determined analyte originated. Further, the amount (oftenexpressed as a concentration and/or quantity) of the unique signatureion, either relative to the amount of other signature ions or relativeto the signature ion associated with a calibration standard (e.g. ananalyte expected in the sample and labeled with a specific reportermoiety), can be used to determine the relative and/or absolute amount(often expressed as a concentration and/or quantity) of analyte in thesample or samples (such as those used to form a sample mixture). In someembodiments, rather than using an internal calibration standard,absolute quantification can be determined based on comparison of thepeak intensities of the various signature ions with a calibration curve.Therefore information, such as the amount of one or more analytes in aparticular sample, can be associated with the reporter moiety that isused to label each particular sample. Where the identity of the analyteor analytes is also determined, that information can be correlated withinformation pertaining to the different signature ions to therebyfacilitate the determination of the identity and amount of each labeledanalyte in one sample or in a plurality of samples.

In some embodiments, the reporter moiety can comprise a nitrogen atomcovalently linked to the methylene carbon of a substituted orunsubstituted N-alkylated acetic acid moiety wherein the substituted orunsubstituted methylene carbon but not the carbonyl group of thecarboxylic acid (or thiocarboxylic acid) group of the acetic acid moietyis part of the reporter. Thus, in some embodiments, the carboxylic acid(or thio carboxylic acid) group can be used to link the reporter to thelinker but it is not considered part of the reporter. The nitrogen atomcan be alkylated with one, two or three groups. For example, the moietycomprising the nitrogen atom can be a substituted primary amine such asa methyl, ethyl or propyl group or a substituted secondary amine such asdimethylamine, diethylamine, di-n-propylamine or diisopropylamine. Thus,for example the reporter moiety “RP” can be illustrated by formulas X¹,X², X³, X⁴, X⁵, X⁶, X⁷, X⁸ or X⁹ as follows wherein the reporter moiety“RP” is set off by the bracket:

The reporter moiety can be a 5, 6 or 7 membered heterocyclic ringcomprising a ring nitrogen atom covalently linked to the methylenecarbon of a substituted or unsubstituted N-alkylated acetic acid moietyto which the analyte is (directly or indirectly) linked through thecarbonyl carbon of the N-alkyl acetic acid moiety and wherein thesubstituted or unsubstituted methylene carbon but not the carbonyl groupof carboxylic acid group is part of the reporter. The heterocyclic ringcan be aromatic or non-aromatic. Thus, the reporter moiety can berepresented by formula Y-J- wherein the group Y can represent the 5, 6or 7 membered heterocyclic ring and the group J can represent thesubstituted or unsubstituted methylene group of the substituted orunsubstituted acetic acid moiety. The heterocyclic ring can besubstituted or unsubstituted. For example, substituents of theheterocylic moiety can include alkyl, alkoxy and/or aryl groups. Thesubstituents can comprise protected or unprotected groups, such asamine, hydroxyl or thiol groups, suitable for linking the analyte to asupport (See FIG. 6). The heterocyclic ring can comprise additionalheteroatoms such as one or more silicon, nitrogen, oxygen and/or sulfuratoms. Thus, for example the reporter moiety “RP” can be illustrated byformulas X¹⁰, X¹¹, X¹², X¹³, X¹⁴, X¹⁵, X¹⁶, X¹⁷, X¹⁸, X¹⁹, X²⁰, X²¹,X²², X²³, X²⁴, X²⁵ or X²⁶ as follows wherein the reporter moiety “RP” isset off by the bracket:

The reporter moiety can be selected so that it does not substantiallysub-fragment under conditions typical for the analysis of the analyte.For the avoidance of any doubt, this is an optional, not a required,feature. For example, the reporter can be chosen so that it does notsubstantially sub-fragment under conditions of dissociative energyapplied to cause fragmentation of the labeled analyte in a massspectrometer. By “does not substantially sub-fragment” we mean thatfragments of the reporter are difficult or impossible to detect abovebackground noise when applied to the successful determination of thelabeled analyte.

In some embodiments, the gross mass of a reporter ion can beintentionally selected to be different as compared with the mass of theanalyte sought to be determined or the mass of any of the expectedfragments of the analyte (i.e. a “quiet zone”). For example, whereproteins or peptides are the analytes, the gross mass of the reporterion can be chosen to be different as compared with any naturallyoccurring amino acid or peptide, or expected fragment ion thereof. Thiscan facilitate analyte determination since, depending on the analyte,the lack of any possible components of the sample having the samecoincident mass can add confidence to the result of any analysis.Examples of mass ranges where little background can be expected forpeptides can be found in Table 1.

TABLE 1 Possible “Quiet Zones” For Selection Of Label Fragment Ion m/zAssociated With Peptide Analysis M/z start-end 10-14 19-22 24-26 31-3840-40 46-50 52-52 58-58 61-69 71-71 74-83 89-97 103-109 113-119 121-125128-128 131-135 137-147 149-154 156-156 160-174 177-182 184-184 188-189191-191 202-207 210-210 216-222 224-226

The reporter moiety can be non-polymeric. The reporter moiety can beselected to produce a signature ion of m/z that indicates its mass isless than 250 atomic mass units (amu). The reporter moiety can beselected to produce a signature ion of m/z less than 200 amu. Thereporter moiety can be selected to produce a signature ion of m/z lessthan 150 amu. Such a small molecule can be easily determined in thesecond mass analysis, free from other components of the sample havingthe same coincident mass in the first mass analysis. In this context,the second mass analysis can be performed, typically in a tandem massspectrometer (or, for example by post source decay in a single stageinstrument), on selected ions that are determined in the first massanalysis. Because ions of a particular mass to charge ratio can bespecifically selected out of the first mass analysis for possiblefragmentation and further mass analysis, the non-selected ions from thefirst mass analysis are not carried forward to the second mass analysisand therefore do not contaminate the spectrum of the second massanalysis. Furthermore, the sensitivity of a mass spectrometer and thelinearity of the detector (for purposes of quantification) can be quiterobust in this low mass range. Additionally, the present state of massspectrometer technology can allow for baseline mass resolution of lessthan one Dalton in this mass range. For all these reasons, reporterspossessing the above described characteristics can provide quiteaccurate quantification of determined analytes from complex mixturesutilizing methods as described herein.

The Balance (or Linker) Moiety:

The balance (or linker) moiety (sometimes referred to by use of theshorthand “LK”) of the labeling reagent or reagents that can be usedwith embodiments of this invention links the reporter moiety to theanalyte or the reporter moiety to the reactive group depending onwhether or not a reaction with the analyte has occurred. The linker canbe selected to produce a neutral species (i.e. undergo neutral loss in amass spectrometer) wherein both the bond that links the linker to thereporter moiety (the RL bond) and the bond that links the linker to theanalyte (the LA bond) fragment in a mass spectrometer. The linker can bedesigned to sub-fragment when subjected to dissociative energy,including sub-fragmentation to thereby produce only neutral fragments ofthe linker. The linker can be designed to produce one or more detectablefragments.

With respect to isobaric sets of reagents, the linker moiety cancomprise one or more heavy atom isotopes such that its mass compensatesfor the difference in gross mass between the reporter moieties for eachlabeled analyte of a mixture or for the labeling reagents of set and/orkit. Moreover, the aggregate gross mass (i.e. the gross mass taken as awhole) of the reporter/linker combination (i.e. the reporter/linkermoiety) can be the same for each labeled analyte of a mixture or for thelabeling reagents of set and/or kit. More specifically, the linkermoiety can compensate for the difference in gross mass between reportermoieties of labeled analytes from different samples (each sample beinglabeled with a different reagent of the isobaric set) wherein the uniquegross mass of the reporter moiety correlates with the sample from whichthe labeled analyte originated and the aggregate gross mass of thereporter/linker combination is the same for each labeled analyte of asample mixture regardless of the sample from which it originated. Inthis way, the gross mass of identical analytes in two or more differentsamples can have the same gross mass when labeled and then mixed toproduce a sample mixture.

For example, the labeled analytes, or the labeling reagents of a setand/or kit for labeling the analytes, can be isobars. Thus, if ions of aparticular mass to charge ratio (taken from the sample mixture) areselected (i.e. selected ions) in a mass spectrometer from an initialmass analysis of the sample mixture, identical analytes from thedifferent samples that make up the sample mixture can be represented inthe selected ions in proportion to their respective concentration and/orquantity in the sample mixture. Accordingly, the linker not only linksthe reporter to the analyte, it also can serve to compensate for thediffering masses of the unique reporter moieties to thereby harmonizethe gross mass of the reporter/linker moiety in the labeled analytes ofthe various samples (c.f. FIG. 4 a and the two sets of isobaric labelingreagents illustrated therein).

Because the linker can act as a mass balance for the reporter moietiesin the labeling reagents greater the number of atoms in the linker, thegreater the possible number of different isomeric/isobaric labelingreagents of a set and/or kit. Stated differently, generally the greaterthe number of atoms that a linker comprises, the greater number ofpotential reporter/linker combinations exist since isotopes can besubstituted at most any position in the linker to thereby produceisomers and/or isobars of the linker portion wherein the linker portionis used to offset the differing masses of the reporter portion andthereby create a set of unique isobaric and/or mass differentiallabeling reagents. Such diverse sets of labeling reagents areparticularly well suited for multiplex analysis of analytes in the sameand/or different samples.

The total number of labeling reagents of a set and/or kit can be two,three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, fifteen, sixteen, seventeen,eighteen, nineteen, twenty or more. The diversity of the labelingreagents of a set or kit is limited only by the number of atoms of thereporter and linker moieties, the heavy atom isotopes available tosubstitute for the light isotopes and the various syntheticconfigurations in which the isotopes can be synthetically placed. Assuggested above however, numerous isotopically enriched basic startingmaterials are readily available from manufacturers such as CambridgeIsotope Laboratories and Isotec. Such isotopically enriched basicstarting materials can be used in the synthetic processes used toproduce sets of isobaric and mass differential labeling reagents (i.e.labeling reagents of different gross mass) or be used to produce theisotopically enriched starting materials that can be used in thesynthetic processes used to produce sets of isobaric and massdifferential labeling reagents.

In some embodiments, the labeling reagents comprise a set of massdifferential tags. For these sets, the linker moiety optionally won'tcomprise any heavy atom isotopes since the linker typically is not beused to ‘balance’ the different reporters. In this way, each differentreagent of the set has a unique mass and a unique reporter moiety thatcan produce a unique signature ion in the mass spectrometer when cleavedfrom the labeled analyte.

Some examples of the preparation of labeling reagents suitable for usein a set of labeling reagents are discussed in more detail below. Forexample, a linker moiety can be represented by formula I^(#);

wherein the atoms or groups represented by X₁, K, R₁ and R₂ aredescribed in more detail below.

The Reporter/Linker Combination (i.e. the Reporter/Linker Moiety):

The labeling reagents can comprise reporter moieties and linker moietiesthat are linked directly to each other. As described above, in someembodiments the reporter/linker moiety can be identical in gross massfor each member of a set and/or kit of labeling reagents (i.e. forisobaric sets of reagents). Moreover, the bond that links the reportermoiety to the linker moiety can be designed to fragment, in at least aportion of the selected ions, when subjected to dissociative energythereby releasing the reporter ion from the linker moiety and/orlinker/analyte moiety (true whether or not the set of reagents isisobaric). Accordingly, the gross mass of the reporter ion (observed asa m/z ratio in the mass spectrometer) and its intensity can be observeddirectly in MS/MS analysis.

The reporter/linker moiety can comprise various combinations of the sameor different heavy atom isotopes amongst the various labeling reagentsof a set or kit. In the scientific literature this has sometimes beenreferred to as “coding”, “isotope coding” or simply as “encoding”. Forexample, Abersold et al. has disclosed the isotope coded affinity tag(ICAT; see WO00/11208). In one respect, the reagents of Abersold et al.differ from some of the labeling reagents of this invention in thatAbersold does not teach two or more same mass labeling reagents such asisomeric and/or isobaric labeling reagents. Rather, Abersold et al.teach about “light” and “heavy” versions of their labeling reagents.With respect to the mass differential labeling reagents disclosedherein, the reagents of Abersold do not fragment in the massspectrometer to release signature ions that can be observed with thefragment (daughter) ions of the analyte. Thus, unlike the Abersoldreagents, the mass differential reagents disclosed herein permitidentification and quantification of an analyte by analysis of a singleMS/MS spectrum or single data set suitable to prepare said spectrum.

In some embodiments, the reporter and/or linker moieties can comprise anatom or group that can be used to immobilize the labeling reagent orlabeled analyte to a support. Immobilization can be direct or indirect.For example, direct immobilization can occur if an atom or group (e.g.an alkyl amine substituent of the reporter and/or linker) associatedwith the reporter and/or linker can, in some embodiments, interactdirectly with a reactive group (e.g. a cleavable linker) of the supportto effect mobilization. By comparison, indirect immobilization occursif, for example, a substituent of the reporter and/or linker (e.g. analkylamine substituent of the reporter and/or linker) is modified (e.g.is biotinylated) and the modifying group interacts with a reactive groupof the support (e.g. avidin or streptavidin) to effect immobilization.Consequently, this invention contemplates embodiments wherein theanalytes can be reacted with support bound labeling reagents whereineach support comprises a unique labeling reagent such that differentsamples are reacted with different supports as well as embodiments whereeach different sample is reacted with a different labeling reagent andthe reaction products are thereafter immobilized to the same or todifferent supports. In either case, a sample mixture is generallyobtained by cleaving the labeled analytes from the support(s) foranalysis by mass spectrometry.

Mass Spectrometers/Mass Spectrometry (MS):

The methods of this invention can be practiced using tandem massspectrometers and other mass spectrometers that have the ability toselect and fragment molecular ions. Tandem mass spectrometers (and to alesser degree single-stage mass spectrometers) have the ability toselect and fragment molecular ions according to their mass-to-charge(m/z) ratio, and then record the resulting fragment (daughter) ionspectra. More specifically, daughter fragment ion spectra can begenerated by subjecting selected ions to dissociative energy (e.g.collision-induced dissociation (CID)). For example, ions correspondingto labeled peptides of a particular m/z ratio can be selected from afirst mass analysis, fragmented and reanalyzed in a second massanalysis. Representative instruments that can perform such tandem massanalysis include, but are not limited to, magnetic four-sector, tandemtime-of-flight, triple quadrupole, ion-trap, and hybrid quadrupoletime-of-flight (Q-TOF) mass spectrometers.

These types of mass spectrometers may be used in conjunction with avariety of ionization sources, including, but not limited to,electrospray ionization (ESI) and matrix-assisted laser desorptionionization (MALDI). Ionization sources can be used to generate chargedspecies for the first mass analysis where the analytes do not alreadypossess a fixed charge. Additional mass spectrometry instruments andfragmentation methods include post-source decay in MALDI-MS instrumentsand high-energy CID using MALDI-TOF (time of flight)-TOF MS. For arecent review of tandem mass spectrometers please see: R. Aebersold andD. Goodlett, Mass Spectrometry in Proteomics. Chem. Rev. 101: 269-295(2001).

Fragmentation by Dissociative Energy:

It is well accepted that bonds can fragment as a result of the processesoccurring in a mass spectrometer. Moreover, bond fragmentation can beinduced in a mass spectrometer by subjecting ions to dissociativeenergy. For example, the dissociative energy can be produced in a massspectrometer by collision-induced dissociation (CID). Other non-limitingexamples of dissociative energy that can be used to fragment ions in amass spectrometer include, but are not limited to, collision activateddissociation (CAD), photoinduced dissociation (PID)), surface induceddissociation (SID)), electron induced dissociation (EID), electroncapture dissociation (ECD)), thermal/black body infrared radiativedissociation (BIRD), post source decay, or combinations thereof. Thoseof ordinary skill in the art of mass spectrometry can appreciate thatother exemplary techniques for imposing dissociative energy that causefragmentation include, but are not limited to, photo dissociation,electron capture and surface induced dissociation.

The process of fragmenting bonds by collision-induced dissociationinvolves increasing the kinetic energy state of selected ions, throughcollision with an inert gas, to a point where bond fragmentation occurs.For example, kinetic energy can be transferred by collision with aninert gas (such as nitrogen, helium or argon) in a collision cell. Theamount of kinetic energy that can be transferred to the ions isproportional to the number of gas molecules that are allowed to enterthe collision cell. When more gas molecules are present, a greateramount of kinetic energy can be transferred to the selected ions, andless kinetic energy is transferred when there are fewer gas moleculespresent.

It is therefore clear that the application of dissociative energy in amass spectrometer can be controlled. It is also well accepted thatcertain bonds are more labile than other bonds. The lability of thebonds in an analyte or the reporter/linker/non-encoded detectable labelmoiety depends upon the nature of the analyte and the nature of thereporter/linker/non-encoded detectable label moiety. Accordingly, thedissociative energy can be adjusted so that the analytes and/or thelabeling reagents (e.g. the reporter/linker combinations) can befragmented in a manner that is determinable. One of skill in the artwill appreciate how to make such routine adjustments to the componentsof a mass spectrometer to thereby achieve the appropriate level ofdissociative energy to thereby fragment at least a portion of ions oflabeled analytes into signature ions (i.e. reporter ions) and daughterfragment ions.

For example, dissociative energy can be applied to ions that areselected/isolated from the first mass analysis. In a tandem massspectrometer, the extracted ions can be subjected to dissociativeenergy, to thereby cause fragmentation, and then transferred to a secondmass analyzer. The selected ions can have a selected mass to chargeratio. The mass to charge ratio can be within a range of mass to chargeratios depending upon the characteristics of the mass spectrometer. Whencollision induced dissociation is used, the ions can be transferred fromthe first to the second mass analyzer by passing them through acollision cell where the dissociative energy can be applied to therebyproduce fragment ions. For example the ions sent to the second massanalyzer for analysis can include some, or a portion, of the remaining(unfragmented) selected ions (if any), as well as reporter ions(signature ions) and daughter fragment ions of the labeled analyte.

Analyte Determination by Computer Assisted Database Analysis:

In some embodiments, analytes can be determined based upon daughter-ionfragmentation patterns that are analyzed by computer-assisted comparisonwith the spectra of known or “theoretical” analytes. For example, thedaughter fragment ion spectrum of a peptide ion fragmented underconditions of low energy CID can be considered the sum of many discretefragmentation events. The common nomenclature differentiates daughterfragment ions according to the amide bond that breaks and the peptidefragment that retains charge following bond fission (Reopstorff et al.,Biomed. Mass Spectrom., 11: 601 (1988)). Charge-retention on theN-terminal side of the fissile amide bond results in the formation of ab-type ion. If the charge remains on the C-terminal side of the brokenamide bond, then the fragment ion is referred to as a y-type ion. Inaddition to b- and y-type ions, the CID mass spectrum may contain otherdiagnostic fragment ions (these include ions generated by neutral lossof ammonia (−17 amu) from glutamine, lysine and arginine or the loss ofwater (−18 amu) from hydroxyl-containing amino acids such as serine andthreonine); the diagnostic fragment ions as well as the b- and y-typeions all being daughter fragment ions. Certain amino acids have beenobserved to fragment more readily under conditions of low-energy CIDthan others. This is particularly apparent for peptides containingproline or aspartic acid residues, and even more so at aspartyl-prolinebonds (Mak, M. et al., Rapid Commun. Mass Spectrom., 12: 837-842(1998)). Accordingly, the peptide bond of a Z′″-pro dimer or Z′″-aspdimer, wherein Z′″ is any natural amino acid, pro is proline and asp isaspartic acid, will tend to be more labile as compared with the peptidebond between all other amino acid dimer combinations.

For peptide and protein samples therefore, low-energy CID spectracontain redundant sequence-specific information in overlapping b- andy-series ions, internal fragment ions from the same peptide, andimmonium and other neutral-loss ions. Interpreting such CID spectra toassemble the amino acid sequence of the parent peptide de novo ischallenging and time-consuming but can be done. Recent advances incomputer assisted de novo methods for sequencing are were described inHuang, Y., Ross, P, Smirnov, I, Martin, S, and Pappin, D. 2003,Proceedings of 6th International Symposium on MS in Health and LifeSciences, Aug. 24-28, 2003, San Francisco Calif. The most significantadvances in identifying peptide sequences have been the development ofcomputer algorithms that correlate peptide CID spectra with peptidesequences that already exist in protein and DNA sequence databases. Suchapproaches are exemplified by programs such as SEQUEST (Eng, J. et al.J. Am. Soc. Mass Spectrom., 5: 976-989 (1994)) and MASCOT (Perkins, D.et al. Electrophoresis, 20: 3551-3567 (1999)).

In brief, experimental peptide CID spectra (MS/MS spectra) are matchedor correlated with ‘theoretical’ daughter fragment ion spectracomputationally generated from peptide sequences obtained from proteinor genome sequence databases. The match or correlation is based upon thesimilarities between the expected mass and the observed mass of thedaughter fragment ions in MS/MS mode. The potential match or correlationis scored according to how well the experimental and ‘theoretical’fragment patterns coincide. The constraints on databases searching for agiven peptide amino acid sequence are so discriminating that a singlepeptide CID spectrum can be adequate for identifying any given proteinin a whole-genome or expressed sequence tag (EST) database. For otherreviews please see: Yates, J. R. Trends, Genetics, 16: 5-8 (2000) andYates, J. R., Electrophoresis 19: 893-900 (1998).

Accordingly, daughter fragment ion analysis of MS/MS spectra can be usednot only to determine the analyte of a labeled analyte, it can also beused to determine analytes from which the determined analyte originated.For example, identification of a peptide in the MS/MS analysis can becan be used to determine the protein from which the peptide was cleavedas a consequence of an enzymatic digestion of the protein. It isenvisioned that such analysis can be applied to other analytes, such asnucleic acids, lipids steroids and/or prostaglandins.

The RL Bond and the LA Bond:

The bond between an atom of the reporter moiety and an atom of thelinker moiety is the RL bond. The bond between an atom of the linkermoiety and an atom of the analyte is the LA bond. In some embodiments,the RL bond and the LA bond can fragment, in at least a portion ofselected ions, when subjected to dissociative energy levels. Therefore,the dissociative energy level can, in some embodiments, be adjusted in amass spectrometer so that both the RL bond and the LA bond fragment inat least a portion of the selected ions of the labeled analytes.

Fragmentation of the RL bond releases the reporter moiety from theanalyte so that the reporter ion can be determined independently fromthe analyte. Fragmentation of LA bond releases the reporter/linkermoiety from the analyte, or the linker from the analyte, depending onwhether or not the RL bond has already fragmented. In some embodiments,the RL bond can be more labile than the LA bond. In some embodiments,the LA bond can be more labile than the RL bond. In some embodiments,the RL and LA bonds can be of the same relative lability. Statedbriefly, the RL bond is designed to fragment to thereby release thereporter ion but the LA bond may, or may not, fragment in the variousembodiments of this invention.

In some embodiments, when the analyte of interest is a protein orpeptide, the relative lability of the RL and LA bonds can be adjustedwith regard to an amide (peptide) bond. The RL bond, the LA bond or bothbonds RL and LA can be more, equal or less labile as compared with atypical amide (peptide) bond. For example, under conditions ofdissociative energy, the RL bond and/or the LA bond can be less prone tofragmentation as compared with the peptide bond of a Z′″-pro dimer orZ′″-asp dimer, wherein Z′″ is any natural amino acid, pro is proline andasp is aspartic acid. In some embodiments, the RL bond and the LA bondcan fragment with approximately the same level of dissociative energy asa typical amide bond. In some embodiments, the RL and LA bonds canfragment at a greater level of dissociative energy as compared with atypical amide bond.

In some embodiments, the RL bond and the LA bond can exist such thatfragmentation of the RL bond results in the fragmentation of the LAbond, and vice versa. In this way, both bonds RL and LA can fragmentessentially simultaneously such that no substantial amount of analyte,or daughter fragment ion thereof, comprises a partial label. By“substantial amount of analyte” it is meant that less than 25%, andpreferably less than 10%, of partially labeled analyte can be determinedin the mass spectrometer (e.g. in MS/MS analysis).

Because in some embodiments there can be a clear demarcation betweenlabeled and unlabeled fragments of the analyte in the mass spectra (e.g.in MS/MS analysis), this feature can simplify the identification of theanalytes from computer assisted analysis of the daughter fragment ionspectra since no compensation for the remnants of the label need beapplied to the mass calculations used to analyze the daughter fragmentions of an analyte. Moreover, because the fragment ions of analytes can,in some embodiments, be either fully labeled or unlabeled (but notpartially labeled), there can be little or no scatter in the masses ofthe daughter fragment ions caused by isotopic distribution acrossfractured bonds such as would be the case where isotopes were present oneach side of a single labile bond of a partially labeled analyteresulting from fragmentation of the labeled analyte caused by theapplication of dissociative energy levels.

The Labeling of Analytes:

As discussed previously, analytes can be labeled by reacting afunctional group of the analyte with the reactive group of the labelingreagent. The functional group on the analyte can be an electrophilicgroup and the functional group of the labeling reagent can be anucleophilic group. The electrophile and nucleophile can react to form acovalent link between the analyte and the labeling reagent.

The labeling reaction can take place in solution. In some embodiments,one of the analyte or the labeling reagent can be support bound. Thelabeling reaction can sometimes be performed in aqueous conditions.Aqueous conditions can be selected for the labeling of biomolecules suchas proteins, peptides and/or nucleic acids. The labeling reaction cansometimes be performed in organic solvent or a mixture of organicsolvents. Organic solvents can be selected for analytes that are smallmolecules. Mixtures of water and organic solvent or organic solvents canbe used across a broad range. For example, a solution of water and fromabout 5 percent to about 95 percent organic solvent or solvents (v/v)can be prepared and used for labeling the analyte. In some embodiments,a solution of water and from about 50 percent to about 95 percentorganic solvent or solvents (v/v) can be prepared and used for labelingthe analyte. In some embodiments, a solution of water and from about 65percent to about 80 percent organic solvent or solvents (v/v) can beprepared and used for labeling the analyte. Non-limiting examples oforganic solvents include N,N′-dimethylformamide (DMF), acetonitrile(ACN), N-Methyl pyrrolidine (NMP) and alcohols such as methanol,ethanol, propanol and/or butanol. Those of skill in the art will be ableto determine appropriate solvent conditions to facilitate analytelabeling depending upon the nature of the labeling reagent and thenature of the analyte using no more than knowledge available in the artand the disclosure provided herein in combination with routineexperimentation.

When performing a labeling reaction, the pH can be modulated. The pH canbe in the range of 4-10. The pH can be outside this range. Generally,the basicity of non-aqueous reactions can be modulated by the additionof non-nucleophilic organic bases. Non-limiting examples of suitablebases include N-methylmorpholine, triethylamine andN,N-diisopropylethylamine. Alternatively, the pH of water containingsolvents can be modulated using biological buffers such as(N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid) (HEPES) or4-morpholineethane-sulfonic acid (MES) or inorganic buffers such assodium carbonate and/or sodium bicarbonate. Because at least one of thereactive groups can be electrophilic, it can be desirable to select thebuffer to not contain any nucleophilic groups. Those of skill in the artwill, with the application of ordinary experimentation, be able toidentify other buffers that can be used to modulate the pH of a labelingreaction so as to facilitate the labeling of an analyte with a labelingreagent. Accordingly, those of skill in the art will be able todetermine appropriate conditions of solvent and pH to thereby facilitateanalyte labeling depending upon the nature of the labeling reagent andthe nature of the analyte using no more than the disclosure providedherein in combination with routine experimentation.

Sample Processing:

In certain embodiments of this invention, a sample can be processedprior to, as well as after, labeling of the analyte or analytes.Processing can facilitate the labeling of the analyte or analytes. Theprocessing can facilitate the analysis of the sample components.Processing can simplify the handling of the samples. Processing canfacilitate two or more of the foregoing.

For example, a sample can be treated with an enzyme or a chemical. Theenzyme can be a protease (to degrade proteins and peptides), a nuclease(to degrade nucleic acids) or some other enzyme. The enzyme can bechosen to have a very predictable degradation pattern. Two or moreproteases and/or two or more nuclease enzymes may also be used together,or with other enzymes, to thereby degrade sample components.

For example, the proteolytic enzyme trypsin is a serine protease thatcleaves peptide bonds between lysine or arginine and an unspecific aminoacid to thereby produce peptides that comprise an amine terminus(N-terminus) and lysine or arginine carboxyl terminal amino acid(C-terminus). In this way the peptides from the cleavage of the proteinare predictable and their presence and/or quantity, in a sample from atrypsin digest, can be indicative of the presence and/or quantity of theprotein of their origin. Moreover, the free amine termini of a peptidecan be a good nucleophile that facilitates its labeling. Other exemplaryproteolytic enzymes include papain, pepsin, ArgC, LysC, V8 protease,AspN, pronase, chymotrypsin and carboxypeptidase (e.g. carboxypeptidaseA, B, C, etc).

For example, a protein (e.g. protein g) might produce three peptides(e.g. peptides B, C and D) when digested with a protease such astrypsin. Accordingly, a sample that has been digested with a proteolyticenzyme, such as trypsin, and that when analyzed is confirmed to containpeptides B, C and D, can be said to have originally comprised theprotein g. The quantity of peptides B, C and D will also correlate withthe quantity of protein g in the sample that was digested. In this way,any determination of the identity and/or quantify of one or more ofpeptides B, C and D in a sample (or a fraction thereof), can be used toidentify and/or quantify protein g in the original sample (or a fractionthereof).

Because activity of the enzymes is predictable, the sequence of peptidesthat are produced from degradation of a protein of known sequence can bepredicted. With this information, “theoretical” peptide information canbe generated. A determination of the ‘theoretical” peptide fragments incomputer assisted analysis of daughter fragment ions (as describedabove) from mass spectrometry analysis of an actual sample can thereforebe used to determine one or more peptides or proteins in one or moreunknown samples (See for example the section above entitled: “AnalyteDetermination By Computer Assisted Database Analysis”).

In some embodiments, sample processing can include treatment ofprecursors to the analyte or analytes to be labeled. For example, if theanalyte or analytes to be labeled are peptides derived from a digestedprotein and the labeling reagent is, for this example, selected to reactthe peptide or peptide analytes, the protein (the analyte precursormolecule) of the sample may be processed in a manner that facilitatesthe labeling reaction. In this example, the protein can be reduced witha reducing agent (e.g. tris[2-carboxyethyl]phosphine (TCEP)) and thethiol groups then blocked by reaction with a blocking reagent (e.g.methyl methanethiosulfonate (MMTS)). In this way the thiol groups of theprotein are blocked and therefore do not interfere with the labelingreaction between the amines of the analytes and labeling reagent.

Those of skill in the art will appreciate that treatment of certainother precursor molecules can be performed using readily availablereagents and protocols that can be adapted with the aid of routingexperimentation. The precise choices or reagents and conditions can beselected depending on the nature of the analyte to be labeled and thelabeling reagent.

In some embodiments, sample processing can include the immobilization ofthe analytes or analyte precursors to a solid support, whether labeledwith a labeling reagent or not. Immobilization can include covalentimmobilization as well as adsorption and other non-covalent means ofimmobilization (e.g. electrostatic immobilization). In some embodiments,immobilization can facilitate reducing sample complexity. In someembodiments, immobilization can facilitate analyte labeling. In someembodiments, immobilization can facilitate analyte precursor labeling.In some embodiments, immobilization can facilitate selective labeling ofa fraction of sample components comprising a certain property (e.g. theycomprise or lack cysteine moieties). In some embodiments, immobilizationcan facilitate purification. The immobilization can facilitate two ormore of the foregoing.

Separation Including Separation of the Sample Mixture:

In some embodiments, the processing of a sample or sample mixture oflabeled analytes can involve separation. One or more separations can beperformed on the labeled or unlabeled analytes, labeled or unlabeledanalyte precursors, or fractions thereof. One or more separations can beperformed on one or more fractions obtained from a solid phase captureor other product of a separations process. Separations can be preformedon two or more of the foregoing.

For example, a sample mixture comprising differentially labeled analytesfrom different samples can be prepared. By differentially labeled wemean that each of the labels comprises a unique property that can beidentified (e.g. comprises a unique reporter moiety that produces aunique “signature ion” in MS/MS analysis). In order to analyze thesample mixture, components of the sample mixture can be separated andmass analysis performed on only a fraction of the sample mixture. Inthis way, the complexity of the analysis can be substantially reducedsince separated analytes can be individually analyzed for mass therebyincreasing the sensitivity of the analysis process. Of course theanalysis can be repeated one or more time on one or more additionalfractions of the sample mixture to thereby allow for the analysis of allfractions of the sample mixture.

Separation conditions under which identical analytes that aredifferentially labeled co-elute at a concentration, or in a quantity,that is in proportion to their abundance in the sample mixture can beused to determine the amount of each labeled analyte in each of thesamples that comprise the sample mixture provided that the amount ofeach sample added to the sample mixture is known. Accordingly, in someembodiments, separation of the sample mixture can simplify the analysiswhilst maintaining the correlation between signals determined in themass analysis (e.g. MS/MS analysis) with the amount of the differentlylabeled analytes in the sample mixture.

The separation can be performed by chromatography. For example, liquidchromatography/mass spectrometry (LC/MS) can be used to effect sampleseparation and mass analysis. Moreover, any chromatographic separationprocess suitable to separate the analytes of interest can be used. Forexample, the chromatographic separation can be normal phasechromatography, reversed-phase chromatography, ion-exchangechromatography (i.e. anion exchange chromatography or cation exchangechromatography), size exclusion chromatography or affinitychromatography.

The separation can be performed electrophoretically. Non-limitingexamples of electrophoretic separations techniques that can be usedinclude, but are not limited to, 1D electrophoretic separation, 2Delectrophoretic separation and/or capillary electrophoretic separation.

An isobaric labeling reagent or a set of reagents can be used to labelthe analytes of a sample. Isobaric labeling reagents are particularlyuseful when a separation step is performed because the isobaric labelsof a set of labeling reagents are essentially indistinguishable (and canbe indistinguishable by gross mass until fragmentation removes thereporter from the analyte). Thus, all analytes of identical compositionthat are labeled with different isobaric labels can chromatograph inexactly the same manner (i.e. co-elute). Because they are essentiallyindistinguishable, the eluent from the separation process can comprisean amount of each isobarically labeled analyte that is in proportion tothe amount of that labeled analyte in the sample mixture. Furthermore,from the knowledge of how the sample mixture was prepared (portions ofsamples and other optional components (e.g. calibration standards) addedto prepare the sample mixture), it is possible to relate the amount oflabeled analyte in the sample mixture back to the amount of that labeledanalyte in the sample from which it originated.

The labeling reagents can also be mass differential labeling reagents(i.e. labeling reagents of different gross mass). Mass differentiallabeling reagents can be used to label, for example, the analytes of twoor more different samples wherein the different labeling reagents of aset all have a distinct mass (i.e. known mass difference as comparedwith other reagents of the set). Because all the reagents of the set canhave a known mass difference, the labeling reagents need not befragmented in order to quantify the relative amounts of like analytes intwo different samples. However, in some embodiments, the labelingreagents can be fragmentable.

Relative and Absolute Quantification of Analytes:

In some embodiments, the relative quantification of differentiallylabeled identical analytes of a sample mixture is possible. For example,relative quantification of differentially labeled identical analytes ispossible by comparison of the relative amounts (e.g. area and/or heightof the peak reported) of reporter ion (i.e. signature ion) that aredetermined in the mass analysis (e.g. in the second mass analysis for aselected, labeled analyte observed in a first mass analysis). Stateddifferently, where each reporter ion can be correlated with informationfor a particular sample used to produce a sample mixture, the relativeamount of that reporter ion, with respect to other reporter ionsobserved in the mass analysis, is the relative amount of that analyte inthe sample mixture. Where components combined to form the sample mixtureare known (e.g. the amount of each sample combined to form a samplemixture), the relative amount of the analyte in each sample used toprepare the sample mixture can be back calculated based upon therelative amounts of reporter ion observed for the labeled analyte ofselected mass to charge. This process can be repeated for all of thedifferent labeled analytes observed in the first mass analysis. In thisway, the relative amount (often expressed in concentration and/orquantity) of each reactive analyte, in each of the different samplesused to produce the sample mixture, can be determined.

In other embodiments, absolute quantification of analytes can bedetermined. For these embodiments, a known amount of one or moredifferentially labeled analytes (the calibration standard or calibrationstandards) can be added to the sample mixture or the intensity of thereporter ion can be correlated with a calibration curve.

A calibration standard can be an expected analyte that is labeled withan isomeric and/or isobaric label of the set of labels used to label theanalytes of the sample mixture provided that the reporter moiety for thecalibration standard is unique as compared with any of the samples usedto form the sample mixture. Once the relative amount of reporter ion forthe calibration standard, or standards, is determined with relation tothe relative amounts of the reporter ion or ions for the differentiallylabeled analytes of the sample mixture, it is possible to calculate theabsolute amount (often expressed in concentration and/or quantity) ofall of the differentially labeled analytes in the sample mixture withreference to the amount of calibration standard or standards that was(were) added to the sample mixture. In this way, the absolute amount ofeach differentially labeled analyte (for which there is a calibrationstandard in the sample from which the analyte originated) can also bedetermined based upon the knowledge of how the sample mixture wasprepared.

Alternatively, a calibration curve can be prepared by analysis ofrepresentative samples of labeled analytes, each sample comprising adifferent known amount of the labeled analyte. The intensities of thepeaks of the reporter ion of the analyzed labeled analyte (isobaricsets) or peaks of labeled analytes (mass differential sets) can beplotted with respect to the known amount of each labeled analyte tothereby generate the standard curve. Once prepared the intensity of areporter ion or labeled analyte (as appropriate) in an unknown samplecan be compared with the standard curve to thereby determine the amountof the analyte in a test sample.

Notwithstanding the foregoing, corrections to the intensity of thereporters ion (signature ions) can be made, as appropriate, for anynaturally occurring, or artificially created, isotopic abundance withinthe reporter moieties. There are numerous ways to correct for isotopicabundance of impurities in the signature ions of reporter moieties. Anexample of such a correction can be found in published copending andco-owned United States Provisional Patent Application No. US2005-0114042 A1, entitled: “Method and Apparatus For De-Convoluting AConvoluted Spectrum”, filed on Aug. 12, 2004. Basically, the intensityof up-mass and down mass peaks associated with the isotopic cluster of asingle labeling reagent can be determined by deconvolution of theconvoluted spectrum of the overlapping isotopic clusters of the labelingreagents using mathematical formulas and calculations. Regardless of howthe values are determined, the more care taken to accurately quantifythe intensity of each reporter ion (i.e. signature ion), the moreaccurate will be the relative and absolute quantification of theanalytes in the original samples.

Proteomic Analysis:

Embodiments of this invention can be used for complex analysis becausesamples can be multiplexed, analyzed and reanalyzed in a rapid andrepetitive manner using mass analysis techniques. For example, samplemixtures can be analyzed for the amount of one or more analytes in oneor more samples. The amount (often expressed in concentration and/orquantity) of the analyte or analytes can be determined for the samplesfrom which the sample mixture was comprised. Because the sampleprocessing and mass analyses can be performed rapidly, these methods canbe repeated numerous times so that the amount of many differentiallylabeled analytes of the sample mixture can be determined with regard totheir relative and/or absolute amounts in the sample from which theanalyte originated.

One application where such a rapid multiplex analysis is useful is inthe area of proteomic analysis. Proteomics can be viewed as anexperimental approach to describe the information encoded in genomicsequences in terms of structure, function and regulation of biologicalprocesses. This may be achieved by systematic analysis of the totalprotein component expressed by a cell or tissue. Mass spectrometry, usedin combination with the method, mixture, kit and/or compositionembodiments of this invention is one possible tool for such globalprotein analysis.

For example, with a set of nine isobaric labeling reagents, it ispossible to obtain nine time points in an experiment to determine up ordown regulation of protein expression, for example, based upon responseof growing cells to a particular stimulant. It is also possible toperform fewer time points but to incorporate one or more controls. It isalso possible to do duplicates or triplicates in the same multiplexexperiment. In all cases, up or down regulation of the proteinexpression, optionally with respect to the one or more optional controlsand/or sample repeats, can be determined in a single multiplexexperiment. Moreover, because processing of the sample mixture isperformed in parallel, the results are directly comparable such that nocompensation need be applied to account for slight variations inprotocol or experimental conditions. Accordingly, experimental analysisfor which these isobaric labeling reagents can be used includes, but isnot limited to, time course experiments, biomarker analysis, multiplexproteomic analysis, mudpit experiments, affinity pull-downs,determination of post-translational modifications (PTMs) and multiplecontrol experiments.

II Compositions

In some embodiments, this invention pertains to compositions representedby formula I;

including a salt form and/or hydrate form thereof; wherein, the groupY-J can be any reporter group. The characteristics of suitable reportergroups have been previously described herein. The characteristics ofsuitable reporter groups have also been described in US Published PatentApplication No. US 2004-0219685-A1 at, inter alia, paragraphs 41-47.

For example, the reporter can comprise a 5, 6 or 7 membered heterocyclicring, as the group Y, wherein said heterocyclic ring may be substitutedor unsubstituted and may optionally be cleavably linked to a support,wherein the heterocyclic ring comprises at least one ring nitrogen atomthat is linked through a covalent bond to the group J. The group J canbe a substituted or unsubstituted methylene group represented by formula—CJ′₂—, wherein each J′ is, independently of the other, hydrogen,deuterium, fluorine, chlorine, bromine, iodine, —R₃, —OR₃, —SR₃, —R₃′OR₃or —R₃′SR₃. The group K can be a group represented by formula: —(CK′₂)—or —((CK′₂)_(m)—X₂—(CK′₂)_(m))_(p)—, wherein n is 0 or an integer from 2to 10, each m is, independently of the other, an integer from 1 to 5, pis an integer from 1 to 4 and each K′ is, independently of the other,hydrogen, deuterium, fluorine, chlorine, bromine, iodine, —R₄, —OR₄,—SR₄, —R₄′OR₄ or —R₄′SR₄. Regarding the groups R₁ and R₂, either: (1) R₁is hydrogen, deuterium or R₆ and R₂ is hydrogen, deuterium or R₇; or (2)R₁ and R₂ taken together is a group represented by formula —(CR′₂)_(q)—or —((CR′₂)_(m)—X₂—(CR′₂)_(m))_(p)— that forms a ring that bridges thetwo nitrogen atoms, wherein q is an integer from 1 to 10, each m is,independently of the other, an integer from 1 to 5, p is an integer from1 to 4 and each R′ is, independently of the other, hydrogen, deuterium,fluorine, chlorine, bromine, iodine, —R₅, —OR₅, —SR₅, —R₅′OR₅ or—R₅′SR₅. The atom or group X₁ can be ═O, ═S, ═NH or ═NR₇. The atom orgroup X₂ can be —O— or —S—. The group Z can be hydrogen or a covalentlylinked analyte. Each R₃, R₄, R₅, R₆ and/or R₇, independently of theother, can be alkyl, alkenyl, alkynyl, aryl, heteroaryl or arylalkyl.For example each R₃, R₄, R₅, R₆ and/or R₇, independently of the other,can be methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butylor tert-butyl. Each R₃′, R₄′ and/or R₅′, independently of the other, canbe alkylene, alkenylene, alkynylene, arylene or alkylarylene. Forexample, each R₃′, R₄′, and/or R₅′, independently of the other, can bemethylene, ethylene, propylene, cyclopropylene, n-butylene,cyclobutylene, n-pentylene, cyclopentylene, n-hexylene or cyclohexylene.

In some embodiments, Y-J can be a group represented by formulas: X¹, X²,X³, X⁴, X⁵, X⁶, X⁷, X⁸ or X⁹ as described above. In some embodiments,Y-J can be a group represented by formulas: X¹⁰, X¹¹, X¹², X¹³, X¹⁴,X¹⁵, X¹⁶, X¹⁷, X¹⁸, X¹⁹ X²⁰, X²¹, X²², X²³, X²⁴, X²⁵ or X²⁶ as describedabove.

The compositions can be isotopically enriched (i.e. encoded). Thecompositions can be isotopically enriched to comprise one or more heavyatom isotopes. The compositions can be isotopically enriched to comprisetwo or more heavy atom isotopes. The compositions can be isotopicallyenriched to comprise three or more heavy atom isotopes. The compositionscan be isotopically enriched to comprise four or more heavy atomisotopes.

The 5, 6 or 7 membered heterocyclic ring can be any 5, 6 or 7 memberedheterocyclic ring that comprises at least one nitrogen atom to which thegroup J can be covalently linked. For example, it can be a substitutedor unsubstituted morpholine, piperidine or piperazine. Possiblesubstituents have been described above in the “Definitions” sectionwherein the heterocyclic ring can comprise one or more of saidsubstituents. For example, a substituent can be hydrogen, deuterium,methyl, —C(H)₂D, —C(H)D₂, —CD₃ or other alkyl (in each case ‘D’ isdeuterium). The substituent can be linked to a heteroatom of the ring.For example, the heterocyclic ring can be N-methylpiperazine. Theheterocyclic ring can be aromatic or non-aromatic.

In some embodiments, the reporter moiety can be cleavably linked to asupport. Various supports are well known in the art. For example,various supports comprising a trityl moiety are sold commercially or canotherwise be prepared (e.g. Trityl chloride support (Trityl-Cl) or2-Chlorotrityl chloride support).

For example, the amino, hydroxyl or thiol group of a reporter moiety ofa labeling reagent can be reacted with the cleavable linker of asuitable support. The cleavable linker can be a “sterically hinderedcleavable linker”. Cleavage of the cleavable linker will release thelabel or a labeled analyte from the support. Non-limiting examples ofsterically hindered solid supports include: Trityl chloride resin(trityl-Cl, Novabiochem, P/N 01-64-0074), 2-Chlorotrityl chloride resin(Novabiochem, P/N 01-64-0021), DHPP (Bachem, P/N Q-1755), MBHA (AppliedBiosystems P/N 400377), 4-methyltrityl chloride resin (Novabiochem, P/N01-64-0075), 4-methoxytrityl chloride resin (Novabiochem, P/N01-64-0076), Hydroxy-(2-chorophnyl)methyl-PS (Novabiochem, P/N01-64-0345), Rink Acid Resin (Novabiochem P/Ns 01-64-0380, 01-64-0202),NovaSyn TGT alcohol resin (Novabiochem, P/N 01-64-0074). Numerous othercleavable linkers are known in the art and can be used to preparesuitable supports using no more than commercially available materials,routine experimentation and the teachings provided herein.

Accordingly, in some embodiments, the 5, 6 or 7 member heterocyclic ringcan comprise an atom or group that facilitates the cleavable linkage ofit to a suitable support. For example, the group can be an alkylene,alkenylene, alkynylene, arylene or alkylarylene group comprising anamino, hydroxyl or thiol group. The atom can be the secondary nitrogenof a piperazine ring. A discussion of exemplary piperazine compounds andmethods for their manufacture can be found in published United Statespatent application No: US 2004-0219685 A1. For example, said supportbound N-alkyl piperazine acetic acid compounds can be reacted with adiamine followed by reaction with a diacid to thereby form support boundcompounds that can be used as labeling reagents, where isotopic encodingis possible based upon the nature of the reactants.

Again with reference to formula I, the group Y-J- (whether or notcleavably linked to a support) can form the reporter moiety. Thereporter moiety can comprise at least one isotopically enriched site.The reporter moiety can comprise at least two isotopically enrichedsites. The reporter moiety can comprise 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16 or 17, or more isotopically enriched sites.

The reporter moiety can either contain a fixed charge or be ionizable ina mass spectrometer. For example, compounds comprising basic groups(e.g. amine groups) are easily protonated to introduce charge and acidiccompounds (e.g. carboxylic acid groups) are easily deprotonated tothereby introduce charge (See: Roth, Kenneth et al, “ChargeDerivatization of Peptides for Analysis by Mass Spectrometry”, MassSpectrometry Reviews, 17: 255-274 (1998)).

The balance (linker) moiety can be formed by the group represented byformula I#;

wherein R₁, R₂, X₁, and K are defined previously. The balance moiety cancomprise at least one isotopically enriched site. The balance moiety cancomprise at least two isotopically enriched sites. The balance moietycan comprise 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17, ormore isotopically enriched sites.

In some embodiments, the composition can be represented by formula II;

including a salt form and/or hydrate form thereof; wherein W is an atomor group that is substituted for at least one M group of the sixmembered heterocyclic ring and is located ortho, meta or para to thenitrogen of the six membered ring. The group W can be —N(H)—, —N(R″)—,—N(R′″)—, —P(R′″)—, —O— or —S—. If selected as —N(R′″)— or —P(R′″)—, thegroup can be used to cleavably link the composition to a support. Eachremaining group M can be, independently of the other, —CM′₂—, whereineach M′ can be, independently of the other, hydrogen, deuterium,fluorine, chlorine, bromine, iodine, —R₈, —OR_(B), —SR₈, —R₈′OR₈ or—R₈′SR₈. The groups J, K, X₁, R₁, R₂ and Z are as previously defined.Each R″, independently of the other, can be alkyl, alkenyl, alkynyl,aryl, heteroaryl or arylalkyl and each R′″ can be H₂N—R₉′—,H(R₁₀)N—R₉′—, (R₁₀)₂N, (R₁₀)₂N—R₉′—, HO—R₉′—, HS—R₉′— or a cleavablelinker that cleavably links the compound to a support. Each R₈ and/orR₁₀ can be, independently of the other, alkyl, alkenyl, alkynyl, aryl,heteroaryl or arylalkyl and each R₈′ and/or R₉′ can be, independently ofthe other, alkylene, alkenylene, alkynylene, arylene or alkylarylene.

In some embodiments, the composition can be represented by formula III;

including a salt form and/or hydrate form thereof, wherein s can be aninteger from 0 to 5. The groups R₁, R₂ and Z are as previously defined.The atom or group R₁₁ can be hydrogen, deuterium, methyl, —C(H)₂D,−C(H)D₂, —CD₃, other alkyl or —R′″, wherein R′″ is as previouslydefined. For example, the composition can be selected from one ofcompounds V-XII or XXV to XXXII as illustrated below.

including a salt form and/or hydrate form thereof; wherein, * indicatesan isotopically enriched site comprising a ¹³C substituted for ¹²C, ¹⁵Nsubstituted for ¹⁴N or ¹⁸O substituted for ¹⁶O, as appropriate and Z ishydrogen or a covalently linked analyte.

In some embodiments, the composition can be represented by formula IV;

including a salt form and/or hydrate form thereof; wherein, R₁₁ and Zare as previously defined. For example, the composition can be selectedfrom one of compounds XV to XXIII or XXXV to XXXXI as illustrated below.

including a salt form and/or hydrate form thereof; wherein, * indicatesan isotopically enriched site comprising a ¹³C substituted for ¹²C, ¹⁵Nsubstituted for ¹⁴N or ¹⁸O substituted for ¹⁶O, as appropriate and Z ishydrogen or a covalently linked analyte.

In some embodiments, the composition can be represented by formula III*;

including a salt form and/or hydrate form thereof; wherein, R₁₁ and Zare as previously defined. For example, the composition can be selectedfrom one of compounds M to MV or MVI to MXIII as illustrated below.

including a salt form and/or hydrate form thereof; wherein, * indicatesan isotopically enriched site comprising a ¹³C substituted for ¹²C, ¹⁵Nsubstituted for ¹⁴N or ¹⁶O substituted for ¹⁶O, as appropriate and Z ishydrogen or a covalently linked analyte.

As stated, the compositions can exist in a salt form and/or hydrateform. Whether or not the composition exists as a salt form willtypically depend upon the nature and number of substituents as well asthe conditions under which it exists and/or was isolated. It is wellknown that basic groups such as amines can be protonated by treatmentwith acid to thereby form salts of the amine. For example, piperazinecontaining labeling reagents can be obtained as a mono-TFA salt, amono-HCl salt, a bis-TFA salt or a bis-HCl salt (See for Example, USPatent Application Publication No. US 2005-0148771 A1). It is also wellknown that acidic groups, such as carboxylic acids, can be deprotonatedby treatment with base to form carboxylate salts. Id. It is alsowell-known that compounds comprising both a basic group such as an amineand an acidic group such as a carboxylic acid can exist in zwitterionicform. All these are considered salt forms and the ionization state ofthese functional groups of the composition will depend either on the pHof any solution in which they exist, or if isolated, on the pH of thesolution from which they were isolated. One of ordinary skill in the artwill surely appreciate how to manipulate the charge state and nature ofany counterion the salt form of the compositions disclosed herein usingno more than routine experimentation and the disclosure provided herein.

Whether or not a composition exists as a hydrate can also depend theconditions under which it exists or was isolated. Hydrates merelycomprise one or more complexed water molecules. This disclosurecontemplates any possible hydrate form or combinations thereof.

As previously described, the group Z can be a covalently linked analyte.Said analytes can be prepared by reaction of the analyte with a labelingreagent. The analyte can be any analyte. For example, the group Z can bea peptide or protein.

The group Z can also be hydrogen and thus comprise a nucleophilicreactive group.

Labeling reagents can organized into sets that can be isobaric and/ormass differential labeling sets. Other properties of the labelingreagents have been disclosed. For example, the labeling reagents can beuseful for the multiplex analysis of one or more analytes in the samesample, or in two or more different samples.

The labeling reagents can be isotopically enriched (i.e. encoded). Thelabeling reagents can be isotopically enriched to comprise one or moreheavy atom isotopes. The labeling reagents can be isotopically enrichedto comprise two or more heavy atom isotopes. The labeling reagents canbe isotopically enriched to comprise three or more heavy atom isotopes.The labeling reagents can be isotopically enriched to comprise four ormore heavy atom isotopes.

In some embodiments, a composition of this invention can be a labeledcalibration standard. As described herein, calibration standards can beadded to mixtures in known quantities to facilitate absolutequantitative analysis of an analyte of interest. Accordingly, in someembodiments, this invention pertains to an analyte, such as a peptide ofinterest, which has been labeled with an isobaric and/or massdifferential labeling reagent. Thus, the labeled calibration standardcan be any analyte labeled with a labeling reagent as described herein.The labeling reagent can be selected from a set of isobaric and/or massdifferential labeling reagents.

III. Methods for Labeling and Analysis

According to some embodiments of this invention, analytes can be labeledand then determined. The labeled analyte, the analyte itself, one ormore fragments of the analyte and/or fragments of the label, can bedetermined by mass analysis. In some embodiments, methods of thisinvention can be used for the analysis of different analytes in the samesample as well as for the multiplex analysis of the same and/ordifferent analytes in two or more different samples. The two or moresamples can be mixed to form a sample mixture. In multiplex analysis,labeling reagents can be used to determine from which sample of a samplemixture an analyte originated. The absolute and/or relative (e.g. withrespect to the same analyte in different samples) amount (oftenexpressed in concentration or quantity) of the analyte, in each of twoor more of the samples combined to form the sample mixture, can bedetermined. Moreover, mass analysis of fragments of the analyte (e.g.daughter fragment ions) can be used to identify the analyte and/or theprecursor to the analyte; such as where the precursor molecule to theanalyte was degraded.

The samples used in the analysis may be any sample comprising analytesthat can be labeled with the labeling reagents disclosed herein such asthose described above under the heading ‘Compositions’. For example, thesample can be a crude or processed cell lysate, a body fluid, a tissueextract or a cell extract. The sample can be a fraction from aseparations process. Other possible sample types have been describedherein.

The analyte in the sample can be any analyte that can be labeled withthe labeling reagent. For example, the analyte can be a peptide and/orprotein. Other possible analyte types have been disclosed herein.

One distinction of the described approach lies in the fact that analytesfrom different samples can be differentially labeled (i.e. encoded) withunique labels that are chemically isobaric (have identical gross mass)or are mass differential tags and that identify the sample from whichthe analyte originated. For isobaric labeling reagents, thedifferentially labeled analytes are not distinguished in MS mode of amass spectrometer because they all have identical (gross) mass to chargeratios. Often, the labeling reagents of a set are selected so that thelabeled analytes are also not distinguishable by separation techniques,such as chromatography or electrophoresis, which might be applied to themixture before the first mass analysis. However, when subjected todissociative energy, such as through collision induced dissociation(CID), the labels can fragment to yield unique reporter ions that can beresolved by mass (mass to charge ratio) in a mass spectrometer. Therelative amount of each unique reporter ion observed in the MS/MS (orMS^(n) wherein n is an integer greater than 1) mass spectrum can becorrelated with the relative amount of a labeled analyte in the samplemixture and, by implication, the relative amount of that analyte in asample from which it originated. Thus, the relative intensities of thereporter ions (i.e. signature ions) can be used to determine therelative amount of an analyte or analytes in two or more differentsamples that were combined to form a sample mixture. From the reporterion information, absolute amounts (often expressed as concentrationand/or quantity) of an analyte or analytes in two or more samples can bederived if calibration standards for each analyte, for which absolutequantification is desired, are incorporated into the sample mixture in aknown quantity or where a calibration curve for the reporter ions orlabeled analytes is available.

For example, the analyte might be a peptide that resulted from thedegradation of a protein using an enzymatic digestion reaction toprocess the sample. Protein degradation can be accomplished by treatmentof the sample with one or more proteolytic enzymes (e.g. trypsin,papain, pepsin, ArgC, LysC, V8 protease, AspN, pronase, chymotrypsin orcarboxypeptidase). By determination of the identity and amount of apeptide in a sample mixture and identifying the sample from which itoriginated, optionally coupled with the determination of other peptidesfrom that sample, the precursor protein to the degraded peptide can beidentified and/or quantified with respect to the sample from which itoriginated. Because this method allows for the multiplex determinationof a protein, or proteins, in more than one sample (i.e. from a samplemixture), it is a multiplex method.

Consequently, in some embodiments, this invention pertains to a methodcomprising reacting two or more samples, each sample comprising one ormore reactive analytes, with a different labeling reagent of a set oflabeling reagents to thereby produce two or more differentially labeledsamples each comprising one or more labeled analytes. The labelingreagents can be selected from a set of isobaric and/or mass differentiallabeling reagents.

For example, the different labeling reagents of the set can berepresented by formula I′;

including a salt form and/or hydrate form thereof, wherein the atoms orgroups Y, J, K, R₁, R₂, and X₁ are as previously defined. In someembodiments, Y can be a substituted or unsubstituted morpholine,piperidine or piperazine moiety.

In some embodiments, the labeling reagents of a set can be isobaricand/or isomeric wherein each different labeling reagent of the set hasthe same gross mass but wherein the group Y-J, which group forms areporter moiety, of each different labeling reagent is uniquely encodedat one or more isotopically enriched sites such that when the bondbetween the group J, of the group Y-J, and the remainder of the labelingreagent fragments in a mass spectrometer, a reporter ion of unique massis produced. In some embodiments, the reporter moiety can comprise asubstituted or unsubstituted piperidine, piperazine or morpholine group.

In some embodiments, the labeling reagents can be a set of massdifferential tags wherein all labels of the set comprise a differentmass. These labeling reagents can be designed to fragment when subjectedto dissociative energy, but need not be so designed since analysis istypically performed in the MS¹ mode. Thus, in some embodiments, thegroup Y-J of each different labeling reagent of the set can be uniquelyencoded at one or more isotopically enriched sites such that when thebond between the group J, of the group Y-J, and the remainder of thelabeling reagent fragments in a mass spectrometer, a reporter ion ofunique mass is produced.

Regardless of whether the set is isobaric (and/or isomeric) or massdifferential, if the labeling reagents are capable of generatingreporter ions, the labeling reagents can, in some embodiments, beselected to each comprise a unique mass. Consequently, each reporter ionof unique mass can be used to identify the sample from which eachlabeled analyte originated.

Reagents of formula I′ comprise a nucleophilic nitrogen which can reactwith a carboxylic acid moiety of the analyte to form an amide. Thenucleophilic nitrogen can also react with an aldehyde or ketone of theanalyte but typically the shifts base is reduced to thereby form astable adduct. Regardless of how formed, the labeled analytes of thesample mixture can be represented by formula I″;

including a salt form and/or hydrate form thereof, wherein the atoms orgroups Y, J, K, R₁, R₂, and X₁ are as previously defined and the groupZ″ can be a covalently linked analyte. In some embodiments, the variableY can be a substituted or unsubstituted morpholine, piperidine orpiperazine group.

The labeling process can produce two or more differentially labeledsamples each comprising one or more labeled analytes. Once the analytesof each sample are labeled with the labeling reagent that is unique tothat sample, the two or more differentially labeled samples, or aportion thereof, can be mixed to produce a sample mixture. The samplemixture can optionally comprise one or more calibration standards.

The volume and/or quantity of each sample combined to produce the samplemixture can be recorded. The volume and/or quantity of each sample,relative to the total sample volume and/or quantity of the samplemixture, can be used to determine a ratio that can be used fordetermining the amount (often expressed in concentration and/orquantity) of an identified analyte in each sample from the analysis ofthe sample mixture. The sample mixture can therefore comprise a complexmixture wherein relative amounts of the same and/or different analytescan be identified and/or quantified, either by relative quantificationof the amounts of analyte in each of the two or more samples orabsolutely where a calibration standard is also added to the samplemixture.

For isobaric sets of labeling reagents, the mixture can, for example, besubjected to spectrometry techniques wherein a first mass analysis canbe performed on the sample mixture, or fraction thereof, using a firstmass analyzer. Ions of a particular mass to charge ratio from the firstmass analysis can then be selected. The selected ions can be subjectedto dissociative energy (e.g. collision-induced dissociation (CID)) tothereby induce fragmentation of the selected ions. By subjecting theselected ions of the labeled analytes to dissociative energy bonds canbe fragmented in at least a portion of the selected ions. In someembodiments, fragmentation of both bonds can cause fragmentation of thereporter/linker moiety as well as cause release the ionized reportermoiety (i.e. the reporter ion or signature ion) from the analyte.Examples of such fragmentation for various labeling reagents isillustrated in FIGS. 1 a to 1 c. Fragmentation of the selected ions bythe dissociative energy can also produce daughter fragment ions of theanalyte. The ions (remaining selected ions, daughter fragment ions andionized reporter moieties (i.e. signature ions)), or a fraction thereof,can then be directed to a second mass analyzer.

In the second mass analyzer, a second mass analysis can be performed onthe selected ions, and the fragments thereof. The second mass analysiscan determine the gross mass (or m/z) and relative amount of each uniquereporter ion that is present at the selected mass to charge ratio aswell as the mass (gross and/or absolute) of some or all of the daughterfragment ions of at least one labeled analyte of the sample mixture. Foreach analyte present at the selected mass to charge ratio, the daughterfragment ions can be used to identify the analyte and/or analytespresent at the selected mass to charge ratio. For example, this analysiscan be done as previously described in the section entitled: “AnalyteDetermination By Computer Assisted Database Analysis”. Thus, in someembodiments, the method further comprises determining the gross mass andrelative amount of each signature ion in the second mass analysis andthe gross and/or absolute mass of some or all of the daughter fragmentions in the second mass analysis. Whether the reagents are from anisobaric set, a mass differential set or a combination of isobaric andmass differential reagents, it is possible to obtain identification andquantification of the analyte by analysis of a single mass spectrum orsingle data set used to produce a mass spectrum since the relevantinformation on reporter ions and daughter fragment ions are present inthe same data set or spectrum. In some embodiments, the method furthercomprises determining the labeled analyte (and/or a precursor thereto)associated with the selected mass to charge ratio by analysis of thedaughter fragment ions.

In some embodiments, certain steps of the process can be repeated one ormore times. For example, in some embodiments, ions of a selected mass tocharge ratio from the first mass spectrometric analysis, different fromany previously selected mass to charge ratio, can be treated todissociative energy to thereby form ionized reporter moieties (i.e.signature ions) and daughter fragment ions of at least some of theselected ions, as previously described. A second mass analysis of theselected ions, the reporter ions and/or the daughter fragment ions, or afraction thereof, can be performed. The gross mass and relative amountof each unique signature ion in the second mass analysis and the mass(gross or absolute) of the daughter fragment ions can also bedetermined. Optionally, the labeled analyte (or precursor molecule)associated with the selected mass to charge ratio can be determined byanalysis of the daughter fragment ions. In this way, the information canbe made available for identifying and/or quantifying one or moreadditional analytes from the first mass analysis.

In some embodiments, it may be useful to repeat the process one or moretimes where the sample mixture has been fractionated (e.g. separated bychromatography or electrophoresis). For example, by repeating theprocess on one or more additional fractions of the sample, it ispossible to analyze the entire sample mixture. It is contemplated thatin some embodiments, the whole process can be repeated one or more timesand within each of these repeats, certain steps can also be repeated oneor more times such as described above. In this way, the contents ofsample mixture can be interrogated and determined to the fullestpossible extent. The entire process can also be repeated on a new set oftwo or more samples.

Those of ordinary skill in the art of mass spectrometry will appreciatethat the first and second mass analysis can be performed in a tandemmass spectrometer. Instruments suitable for performing tandem massanalysis have been previously described herein. Although tandem massspectrometers are preferred, single-stage mass spectrometers may beused. For example, analyte fragmentation may be induced by cone-voltagefragmentation, followed by mass analysis of the resulting fragmentsusing a single-stage quadrupole or time-of-flight mass spectrometer. Inother examples, analytes may be subjected to dissociative energy using alaser source and the resulting fragments recorded following post-sourcedecay in time-of-flight or tandem time-of-flight (TOF-TOF) massspectrometers.

As previously discussed, in some embodiments, the labeling reagents orthe set of labeling reagents can be support bound. Accordingly, exceptfor accommodating recapture of the labeled analytes from the support,the above described methods can be practiced with the support boundreagents. This, in some embodiments, this invention pertains topracticing any of the above disclosed methods, wherein each differentlabeling reagent of the set is support bound and is linked to thesupport through a cleavable linker such that each different sample isreacted with a support carrying a different labeling reagent of the setand wherein the method further comprises, after performing the step oflabeling the sample but before performing the step of mixing the labeledsamples to prepare the sample mixture: i) optionally washing eachsupport to remove components of each sample that do not react with thereactive group of the support bound labeling reagent; ii) cleaving thecleavable linker to thereby release the labeled analytes from eachsupport, each differentially labeled sample comprising one or morelabeled analytes wherein the labeled analytes associated with aparticular sample are identifiable and/or quantifiable by the reportermoiety of unique mass linked thereto; and iii) optionally collecting thelabeled analytes of each sample prior to mixing them.

In some embodiments, methods of the invention can further comprisedigesting each sample with at least one enzyme to partially, or fully,degrade components of the sample prior to performing the labeling of theanalytes of the sample (Also see the above section entitled: “SampleProcessing”). For example, the enzyme can be a protease (to degradeproteins and/or peptides) or a nuclease (to degrade nucleic acids). Twoor more enzymes may also be used together to thereby further degradesample components. For example, the enzyme can be a proteolytic enzymesuch as trypsin, papain, pepsin, ArgC, LysC, V8 protease, AspN, pronase,chymotrypsin or a carboxypeptidase (e.g. A, B, C, etc).

In some embodiments, methods can further comprise separating the samplemixture prior to performing the first mass analysis (Also see the abovesection entitled: “Separation Including Separation Of The SampleMixture”). In this manner the first mass analysis can be performed ononly a fraction of the sample mixture. The separation can be performedby any separations method, including by chromatography and/or byelectrophoresis. For example, liquid chromatography/mass spectrometry(LC/MS) can be used to effect such a sample separation prior to the massanalysis. Moreover, any chromatographic separation process suitable toseparate the analytes of interest can be used. Non-limiting examples ofsuitable chromatographic and electrophoretic separations processes havebeen described herein.

In some embodiments, the methods can be practiced with digestion andseparation steps. While these steps are optional, they often areperformed together, for example, when proteomic analysis is being doneto thereby determine the up and down regulation of proteins in cells. Insome embodiments, the steps of the methods, with or without thedigestion and/or separation steps, can be repeated one or more times tothereby identify and/or quantify one or more other analytes in a sampleor one or more analytes in each of the two or more samples (includingsamples labeled with support bound labeling reagents). Depending ofwhether or not a calibration standard is present in the sample mixture,the quantification of a particular analyte can be relative to the otherlabeled analytes, or it can be absolute.

As described previously, it is possible to determine the analyteassociated with the selected ions by analysis of the mass (gross orabsolute) of the daughter fragment ions. One such method ofdetermination is described in the section entitled: “AnalyteDetermination By Computer Assisted Database Analysis”. Once the analytehas been determined, information regarding the gross mass and relativeamount of each unique reporter ion in the second mass analysis and themass of daughter fragment ions provides the basis to determine otherinformation about the sample mixture.

The relative amount of reporter ion can be determined by peak intensityin the mass spectrum. In some embodiments, the amount of each uniquereporter ion can be determined by analysis of the peak height or peakwidth (or peak area) of the reporter ion (signature ion) obtained usinga mass spectrometer. Because each sample can be labeled with a differentlabeling reagent and each labeling reagent can comprise a uniquereporter moiety that produces a unique reporter ion that can becorrelated with a particular differentially labeled sample used toformulate the sample mixture, determination of the different reporterions in the second mass analysis can be used to identify thedifferentially labeled sample from which the reporter ions of theselected analyte originated. Where multiple reporter ions are found(e.g. according to the multiplex methods of the invention), the relativeamount of each unique reporter ion can be determined with respect to theother reporter ions. Because the relative amount of each unique reporterion determined in the second mass analysis can be correlated with therelative amount of an analyte in the sample mixture and because theratios of samples used to prepare the sample mixture can be known, therelative amount (often expressed as concentration and/or quantity) ofthe analyte in each of the differentially labeled samples combined toform the sample mixture can be determined. Moreover, it is possible torelate the quantification information for an analyte to components ofthe original differentially labeled samples where an analyte that isdetermined is a by-product from another compound of interest (e.g. theanalyte is a product of a degradation reaction such as where the analyteis a peptide formed by the digestion of a protein).

As discussed above, this analysis can be repeated one or more times onselected ions of a different mass to charge ratio to thereby obtain therelative amount of one or more additional analytes in each samplecombined to form the sample mixture. Moreover, as appropriate, acorrection of peak intensity associated with each unique reporter ioncan be performed for naturally occurring, or artificially created,isotopic abundance, as previously discussed in the section entitled:“Relative and Absolute Quantification of Analytes”.

In some embodiments, the analytes can be peptides in a sample or samplemixture. Analysis of the peptides in a sample, or sample mixture, can beused to determine the amount (often expressed as a concentration and/orquantity) of identifiable proteins in the sample or sample mixturewherein proteins in one or more samples can be degraded prior to thefirst mass analysis. Moreover, the information from different samplescan be compared for the purpose of making determinations, such as forthe comparison of the effect on the amount of the protein in cells thatare incubated with differing concentrations of a substance that mayaffect cell growth, development, differentiation and/or death. Other,non-limiting examples may include comparison of the expressed proteincomponents of diseased and healthy tissue or cell cultures. This mayencompass comparison of expressed protein levels in cells, tissues orbiological fluids following infection with an infective agent such as abacteria or virus or other disease states such as cancer. In otherexamples, changes in protein concentration over time (time-course)studies may be undertaken to examine the effect of drug treatment on theexpressed protein component of cells or tissues. In still otherexamples, the information from different samples taken over time may beused to detect and monitor the concentration of specific proteins intissues, organs or biological fluids as a result of disease (e.g.cancer) or infection. Such experiments may include one or more controlsamples. In some embodiments, the experiments can be used to determinetwo or more of the characteristics of interest described above.

Where a calibration standard comprising a unique reporter moiety islinked to an analyte, having the selected mass to charge ratio, has beenadded to the sample mixture in a known amount (often expressed as aconcentration and/or quantity), the amount of the unique reporterassociated with the calibration standard can be used to determine theabsolute amount (often expressed as a concentration and/or quantity) ofthe analyte in each of the samples combined to form the sample mixture.This is possible because the amount of analyte associated with theunique reporter ion for the calibration standard in the sample mixtureis known and the relative amounts of all unique reporter ions can bedetermined for the labeled analyte associated with the selected ions.Since the relative amount of each unique reporter ion, determined foreach of the unique reporters moieties (including the reporter moiety forthe calibration standard), is proportional to the amount of the analyteassociated with each differentially labeled sample combined to form thesample mixture, the absolute amount (often expressed as a concentrationand/or quantity) of the analyte in each of the samples can be determinedbased upon a ratio calculated with respect to the formulation used toproduce the sample mixture. As appropriate, a correction of peakintensity associated with each of the unique reporter ions can beperformed for naturally occurring, or artificially created, isotopicabundance. Such an analysis method can be particularly useful forproteomic analysis of multiplex samples of a complex nature, especiallywhere a preliminary separation of the labeled analytes (e.g. liquidchromatography or electrophoretic separation) precedes the first massanalysis.

For example, if a sample mixture comprises 100 fmol/mL of a calibrationstandard and the relative intensity of the unique reporter ionassociated with the calibration standard was 1 while the relativeintensity of a first other unique reporter ion associated with a firstsample was one-half and the relative intensity of a second other uniquereporter ion associated with a second sample was 2, the amount of theanalyte in the first differentially labeled sample mixed to form thesample mixture (assuming equal amounts of sample 1 and sample 2 aremixed to form the sample mixture) is 50 fmol/mL (0.5×100 fmol/mL) andthe amount of the analyte in the second differentially labeled samplemixed to form the sample mixture is 200 fmol/mL (2×100 fmol/mL).Moreover, if, for example, the analyte is a peptide associated with aparticular protein, it can be inferred that the amount of the protein insample 1 is 50 fmol/mL and the amount of the protein in sample 2 is 200fmol/mL. Thus, the presence of the calibration standard permits absolutequantification of the labeled analytes (and in some cases theirprecursors) in each differentially labeled sample mixed to form thesample mixture.

As previously discussed, in some embodiments, the absolute amount ofeach signature ion of unique mass, that corresponds to each uniquereporter moiety, can be determined with reference to a calibrationcurve. Accordingly, the absolute amount of the determined analyte ineach different sample of the sample mixture can be determined withreference to the absolute amount of each different signature ion ofunique mass.

As previously discussed, this analysis can be repeated one or more timeson selected ions of a different mass to charge ratio to thereby obtainthe absolute amount of one or more additional analytes in each samplecombined to form the sample mixture. Moreover, as appropriate, acorrection of peak intensity associated with each unique reporter ioncan be performed for naturally occurring, or artificially created,isotopic abundance, as previously discussed.

In some embodiments, methods described herein can be practiced using alabeling reagent that can be represented by formula II′;

including a salt form and/or hydrate form thereof; wherein W, M, J, K,R₁, R₂ and X₁ are as previously described.

In some embodiments, methods described herein can be practiced using atleast one labeling reagent that can be represented by formula III′:

including a salt form and/or hydrate form thereof, wherein s, R₁, R₂,and R₁₁ are as previously described. For example, the method can bepracticed using at least one labeling reagent represented by formula V′to XII′ as illustrated below:

including a salt form and/or hydrate form thereof, wherein, R₁, and R₂are as previously described and the symbol * indicates an isotopicallyenriched site comprising a ¹³C substituted for ¹²C, ¹⁵N substituted for¹⁴N or ¹⁸O substituted for ¹⁶O, as appropriate.

In some embodiments, the method can be practiced using at least onelabeling reagent that can be represented by formula IV′;

including a salt form and/or hydrate form thereof, wherein R₁₁ is aspreviously described. For example, the method can be practiced using atleast one labeling reagent that can be represented by formula XV′ toXXIII′ as illustrated below;

including a salt form and/or hydrate form thereof wherein, * indicatesan isotopically enriched site comprising a ¹³C substituted for ¹²C, ¹⁵Nsubstituted for ¹⁴N or ¹⁶O substituted for ¹⁶O, as appropriate.

In some embodiments, the method can be practiced using at least onelabeling reagent that can be represented by formula III*′;

including a salt form and/or hydrate form thereof, wherein R₁₁ is aspreviously described. For example, the method can be practiced using atleast one labeling reagent that can be represented by formula M′ to MV′as illustrated below;

including a salt form and/or hydrate form thereof, wherein, * indicatesan isotopically enriched site comprising a ¹³C substituted for ¹²C, ¹⁵Nsubstituted for ¹⁴N or ¹⁸O substituted for ¹⁸O, as appropriate.

As discussed previously, in some embodiments the method can be performedusing sets of mass differential tags. Therefore, in some embodiments,the method can further comprise performing a first mass spectrometricanalysis on the sample mixture, or a fraction thereof, and determiningthe relative intensity of peaks associated with labeled analytes. Sincethe identity of the analytes can be determined by analysis of daughterion fragments, the method can further comprising fragmenting ions of thelabeled analytes to dissociative energy to thereby form daughter ionfragments and identifying the analyte from the daughter ion fragments.

In some embodiments, this method can be practiced using at least onelabeling reagent that can be represented by formula XXV′ to XXXII′ asillustrated below;

including a salt form and/or hydrate form thereof, wherein, * indicatesan isotopically enriched site comprising a ¹³C substituted for ¹²C, ¹⁵Nsubstituted for ¹⁴N or ¹⁸O substituted for ¹⁶O, as appropriate.

In some embodiments, this method can be practiced using at least onelabeling reagent that can be represented by formula XXXV′ to XXXXI′ asillustrated below;

including a salt form and/or hydrate form thereof, wherein, * indicatesan isotopically enriched site comprising a ¹³C substituted for ¹²C, ¹⁵Nsubstituted for ¹⁴N or ¹⁸O substituted for ¹⁶O, as appropriate.

In some embodiments, this method can be practiced using at least onelabeling reagent that can be represented by formula MVI′ to MXIII′ asillustrated below;

including a salt form and/or hydrate form thereof, wherein, * indicatesan isotopically enriched site comprising a ¹³C substituted for ¹²O, ¹⁵Nsubstituted for ¹⁴N or ¹⁸O substituted for ¹⁶O, as appropriate.Proteomic Workflows

In some embodiments, the labeling of the analytes of a sample can beperformed prior to performing sample processing steps. In someembodiments, the labeling of analytes can be performed amongst othersample processing steps. In some embodiments, the labeling of analytesis the last step of sample processing and/or immediately precedes thepreparation of a sample mixture.

Using proteomic analysis as a non-limiting example, there are at leastseveral possible workflows that might be used. To aid in understandingof the following discussion a distinction is sometimes made between theprecursor protein and the analyte peptide. However, it should beunderstood that in various embodiments either, or both, proteins and/orpeptides could be considered analytes as described herein.

In one type of workflow, the precursor proteins can be digested topeptide analytes that can thereafter be labeled. In another type ofworkflow, the precursor proteins can be labeled with the labelingreagent and then digested to labeled peptide analytes. In another typeof workflow, the precursor proteins can be captured on a solid support,digested and then the support bound peptides can be labeled. Optionallythe flow through peptides can also be labeled. In another type ofworkflow, the precursor proteins can be captured on a solid support,labeled and then the support bound protein can be digested to producelabeled peptides. Optionally the flow through peptides can also beanalyzed. Regardless of the workflow, additional sample processing (e.g.separation steps) can be performed on the labeled peptides as desiredbefore MS and MS/MS analysis.

Exemplary Workflows Involving Digestion Followed by Labeling

As an example, there may be a “control” sample and a “test” sample to beanalyzed. If, for the example, the goal is to analyze peptides (as theanalytes) of “control” and “test” sample proteins, the proteins of thesamples can, in some embodiments, be optionally reduced, optionallycysteine blocked and digested with an enzyme to thereby produce theanalyte peptides that can be labeled for subsequent analysis. Theanalyte peptides can, in some embodiments, be labeled (tagged) withoutfurther sample processing. Regardless of how labeled, the analytes ofeach different sample can be labeled with using different labelingreagents each comprising a reporter moiety of unique mass (e.g. thelabeling reagents of a set of isomeric and/or isobaric labels).

In some embodiments, further sample processing might be desired beforelabeling and/or after labeling. For example, a separation step might beperformed to eliminate certain types of peptides that are not ofinterest, thereby decreasing the complexity of the sample. The labeledsamples can be mixed to obtain a sample mixture. In some embodiments,the labeled analyte peptide can be subject to separation (e.g. highperformance liquid chromatography (HPLC)) before mass spectral analysis.

Another exemplary embodiment includes optional steps of blocking andregeneration of the thiol groups of cysteine that can be involved withpeptide capture. For the avoidance of doubt, it is self-evident thatadditional samples can be processed provided that additionaldifferential labels are available to encode each different sample orsample fraction.

In some embodiments, the “control” sample and the “test” samples can bedigested with an enzyme and then components of the sample can becaptured on a solid phase through a cleavable linker. For example, thesupport can comprise a cleavable linker and a reactive group that reactswith moieties of a peptide.

In some embodiments, the peptides that flow through the support (becausethey do not react with the functional group of the support) can (insteadof being discarded) be collected, labeled with a labeling reagent of aset of isobaric and/or mass differential labeling reagents and beanalyzed separately or together with the labeled peptides collected fromthe support. The peptides that flow through the solid support can belabeled with the same or with a different labeling reagent of a set oflabeling reagents. Regardless of the labeling reagent, they canoptionally be mixed with the sample mixture that is analyzed by MS/MSanalysis. They also can be independently analyzed. It is possible tolabel the peptides retained on the support either while still on thesupport or after they have been cleaved from the support.

It is also possible to use a solid support to capture the precursorproteins. For example, there can be two samples processed using aparallel path. The proteins that do not comprise a cysteine moiety canbe removed from the support with a wash and optionally be collected(i.e. flow through). They can also be optionally digested, labeledand/or analyzed with the sample mixture or be analyzed separately.

The support bound proteins can be digested. The support bound cysteinecomprising peptides can then be labeled with labeling reagent andcleaved from the support. The support bound cysteine comprising peptidescan otherwise first be cleaved from the support and then labeled withlabeling reagent. Labeled peptides from the different samples(optionally including the labeled peptides that do not comprise cysteinemoieties) can be mixed, processed and/or analyzed with the samplemixture or be analyzed separately.

It is also possible to collect any peptides that are released from thesupport as a consequence of performing the digestion. Typically theseare peptides that do not comprise a thiol group. These peptides canoptionally be labeled with a labeling reagent and optionally mixed,processed and/or analyzed with the sample mixture or be analyzedseparately.

Exemplary Workflows Involving Labeling Followed by Digestion

Whether or not a support is used to capture an analyte for analysis, thestep of labeling the analyte with a labeling reagent can be performedeither before or after digestion or other chemical treatment providedthat the treatment does not modify the label in a way that would renderit non-operative for quantifying labeled analytes as described herein.For protein samples, it is also possible to reduce and cysteine blockthe sample protein, label the N-ε-lysine side chain amine groups of thesample protein with the labeling reagent and then digest the proteininto labeled peptides.

Regardless of their origin, labeled analytes can be analyzed or they canbe further processed (including preparing a sample mixture), for exampleby separation and/or by immobilization to a support. The labeled proteincan be cleaved from the support and then digested or the labeled proteincan be digested while still support bound. In the latter case, supportbound digestion will free peptides from the support that do not comprisea cysteine moiety. These can be collected and optionally analyzed eitherseparately or as part of the sample mixture comprising the laterreleased labeled peptides comprising cysteine moieties.

When the precursor proteins are labeled before digestion to peptides,the digestion pattern can be altered. For example, digestion withtrypsin can be expected to produce predominately C-terminal argininepeptides because the N-ε-lysine side chain amine groups are modifiedwith the label. Consequently, the activity of trypsin can be much likethat of Arg-C. Because only those C-terminal arginine peptides that alsocomprise a lysine side chain can be labeled and therefore detectable inthe mass spectrometer, this offers a way to further reduce thecomplexity of the sample to be further processed and/or analyzed.

In some embodiments, it is possible to reduce the protein and label thecysteine groups with labeling reagent (i.e. a thiol specific labelingreagent) and then digest the protein into labeled peptides for analysis.The labeled peptide analytes can be analyzed or can be furtherprocessed, for example by separation and/or immobilization to a support.For example, it is possible to immobilize labeled peptides to a supportby reaction of the N-α-amine groups and/or the N-ε-amine groups of thelysine side chains with functional groups of the support. Supports withcleavable linkers for the immobilization of compounds comprising aminefunctional groups include supports comprising trityl linkers (See:Trityl chloride support (Trityl-Cl) or 2-Chlorotrityl chloride supportavailable from Novabiochem (San Diego, Calif.)). This workflow isdistinct from those described previously. The labeled analytes can becleaved from the support, further processed and/or analyzed. Thisprocess might not provide substantial complexity reduction since all ofthe digested peptides are expected to comprise at least an N-α-aminegroup.

The foregoing examples are not intended to be exhaustive of variouspossible workflows. They are intended to be exemplary only. With regardto embodiments where labeling precedes digestion, it is also possible toengage in further sample processing prior to performing the digestion.

Summary

Whilst the preceding discussion focused, by way of specific example, onproteomic analysis and the determination of peptides and/or proteins asanalytes, the concepts described are intended to encompass many types ofanalytes for which the preceding workflows are applicable without theexercise of undue experimentation. Accordingly, the scope of thisdisclosure is not intended to be limited to any of these specificexamples discussed.

IV. Mixtures

In some embodiments, this invention pertains to mixtures (i.e. samplemixtures). For example, the mixtures can comprise isobarically and/ormass differential labeled analytes. Exemplary mixtures of labeledanalytes and methods for their preparation and/or analysis have beendescribed in the section entitled “Methods for Labeling and Analysis”,set forth above.

The mixture can be formed by mixing all, or a part, of the product oftwo or more labeling reactions wherein each sample is labeled with adifferent labeling reagent of a set of labeling reagent wherein eachlabeling reagent comprises a reporter moiety of unique (gross) mass. Theunique reporter moiety of each different labeling reagent can identifyfrom which labeling reaction each of the two or more labeled analytes isderived (i.e. originated). The labeling reagents can be isotopicallyencoded isobaric (and/or isomeric) and/or mass differential labelingreagents. Hence, two or more of the labeled analytes of a mixture can beisobaric (and/or isomeric) and/or mass differential. Characteristics ofthe labeling reagents and labeled analytes associated with those methodshave been previously discussed.

The analytes of the mixture can be any analyte. For example, theanalytes of the mixture can be peptides. The analytes of the mixture canbe proteins. The analytes of the mixture can be peptides and proteins.The analytes of the mixture can be nucleic acid molecules. The analytesof the mixture can be carbohydrates. The analytes of the mixture can belipids. The analytes of the mixture can be prostaglandins, the analytesof the mixture can be fatty acids. The analytes of the mixture can becarnitines. The analytes of the mixture can be amino acids. The analytesof the mixture can be vitamins. The analytes of the mixture can besteroids. The analytes of the mixture can be small molecules having amass of less than 1500 daltons. The analytes of the mixture comprise twoor more different analyte types (e.g. 1) lipids and steroids; or 2)peptides, lipids, steroids and carbohydrates).

Mixtures can comprise any type of differentially labeled analytescomprising novel reporter/linker moiety disclosed herein. For example,the mixtures can comprise at least two differentially labeled analytesthat can be represented by formula I″;

including a salt form and/or hydrate form thereof, wherein the atoms orgroups Y, J, K, R₁, R₂, and X₁ have been previously described and theircharacteristics disclosed and wherein the group represented by formulaI^;

is the same for all labeled analytes associated with a particular samplebut wherein the group of formula I^ comprises at least one isotopicallyenriched site that differs from that of any other labeled analyte thatoriginates from a different sample combined to form the mixture. Z″ isthe covalently linked analyte.

In some embodiments, each of the two-labeled analytes can originate froma different sample. In some embodiments the group Y-J formula I″, whichgroup can form a reporter moiety, of each different labeled analyte canbe uniquely encoded at one or more isotopically enriched sites such thatwhen the bond between the group J, of the group Y-J, and the remainderof the labeled analyte fragments in a mass spectrometer, a reporter ionof unique mass is produced that is different from any reporter ionassociated with any other labeled analyte that originates from adifferent sample combined to form the mixture and wherein said uniquereporter ion is capable of identifying the sample from which the labeledanalyte originated. The group Z″ can be a covalently linked analyte. Foreach different label, some of the labeled analytes of the mixture can bethe same and some of the labeled analytes can be different.

In some embodiments, the group represented by formula I^;

comprises a gross mass that is the same for all labeled analytesassociated with a particular sample but that is different in gross massfrom group of formula I^ associated with any other labeled analyte thatoriginates from a different sample combined to form the mixture andwherein the gross mass of the group of formula I^ is capable ofidentifying the sample from which the labeled analyte originated.

In some embodiments, the mixture can comprise at least twodifferentially labeled analytes that can be represented by formula II″;

including a salt form and/or hydrate form thereof; wherein W, M, J, K,R₁, R₂, X₁, and Z″ are as previously described.

In some embodiments, the mixture can comprise at least twodifferentially labeled analytes wherein at least one labeled analyte isrepresented by formula III″;

including a salt form and/or hydrate form thereof, wherein s, R₁, R₂,R₁₁ and Z″ are as previously described.

In some embodiments, the mixture can comprise at least twodifferentially labeled analytes wherein at least one labeled analyte isrepresented by formula V″, VI″, VII″, VIII″, IX″, X″, XI″ or XII″;

including a salt form and/or hydrate form thereof, wherein, * indicatesan isotopically enriched site comprising a ¹³C substituted for ¹²C, ¹⁵Nsubstituted for ¹⁴N or ¹⁸O substituted for ¹⁶O, as appropriate.

In some embodiments, the mixture can comprise at least twodifferentially labeled analytes wherein at least one labeled analyte isrepresented by formula XXV″, XXVI″, XXVII″, XXVIII″, XXIX″, XXX″, XXXI″or XXXII″;

including a salt form and/or hydrate form thereof, wherein, * indicatesan isotopically enriched site comprising a ¹³C substituted for ¹²C, ¹⁵Nsubstituted for ¹⁴N or ¹⁸O substituted for ¹⁶O, as appropriate.

In some embodiments, the mixture can comprise at least twodifferentially labeled analytes wherein at least one labeled analyte isrepresented by formula IV″;

including a salt form and/or hydrate form thereof, wherein R₁₁ and Z″are as previously described.

In some embodiments, the mixture can comprise at least twodifferentially labeled analytes wherein at least one labeled analyte isrepresented by formula XV″, XVI″, XVII″, XVIII″, XIX″, XX″, XXI″, XXII″or XXIII″:

including a salt form and/or hydrate form thereof, wherein, * indicatesan isotopically enriched site comprising a ¹³C substituted for ¹²C, ¹⁵Nsubstituted for ¹⁴N or ¹⁵O substituted for ¹⁶O, as appropriate.

In some embodiments, the mixture can comprise at least twodifferentially labeled analytes wherein at least one labeled analyte isrepresented by formula XXXV″, XXXVI″, XXXVII″, XXXVIII″, XXXIX″, XXXX″or XXXXI″:

including a salt form and/or hydrate form thereof, wherein, * indicatesan isotopically enriched site comprising a ¹³C substituted for ¹²C, ¹⁵Nsubstituted for ¹⁴N or ¹⁸O substituted for ¹⁶O, as appropriate.

In some embodiments, the mixture can comprise at least twodifferentially labeled analytes wherein at least one labeled analyte isrepresented by formula III*″;

including a salt form and/or hydrate form thereof, wherein R₁₁ and Z″are as previously described.

In some embodiments, the mixture can comprise at least twodifferentially labeled analytes wherein at least one labeled analyte isrepresented by formula M″, MI″, MII″, MIII″, MIV″ or MV″:

including a salt form and/or hydrate form thereof, wherein, * indicatesan isotopically enriched site comprising a ¹³C substituted for ¹²C, ¹⁵Nsubstituted for ¹⁴N or ¹⁸O substituted for ¹⁶O, as appropriate.

In some embodiments, the mixture can comprise at least twodifferentially labeled analytes wherein at least one labeled analyte isrepresented by formula MVI″, MVII″, MVIII″, MIX″, MX″, MXI″, MXII″ orMXIII″:

including a salt form and/or hydrate form thereof, wherein, w indicatesan isotopically enriched site comprising a ¹³C substituted for ¹²C, ¹⁵Nsubstituted for ¹⁴N or ¹⁸O substituted for ¹⁶O, as appropriate.V. Kits

In some embodiments, this invention pertains to kits. The kits cancomprise a labeling reagent as described herein and one or more otherreagents, containers, enzymes, buffers and/or instructions. The kits cancomprise a set of two or more labeling reagents and one or more otherreagents, containers, enzymes, buffers and/or instructions. For example,the kit can comprise at least one additional reagent selected to performan assay for quantifying one or more analytes in two or more differentsamples. For example, the kit can comprise a labeled calibrationstandard comprising a reporter moiety of unique gross mass. For example,the kit can comprise a labeled calibration standard comprising a labelmoiety of unique gross mass.

Two or more of the labeling reagents of a kit can be isomeric and/orisobaric. For example, one or more labeling reagents of the kits can becompounds (including sets of compounds) of the formula: I′, II′, III′,IV′, V′, VI′, VII′, VIII′, IX′, X′, XI′, XII′, XIII′, XV′, XVI′, XVII′,XVIII′, XIX′, XX′, XXI′, XXII′ and/or XXIII′, as previously disclosedherein. In some embodiments, the kit can comprise a labeled analyte (forexample as a calibration standard) of formula: I″, II″, III″, IV″, V″,VI″, VII″, VIII″, IX″, X″, XI″, XII″, XIII″, XV″, XVI″, XVII″, XVIII″,XIX″, XX″, XXI″, XXII″ and/or XXIII″, as previously disclosed herein.Other properties of the labeling reagents of the kits have beendisclosed. The kits can, for example, be useful for the multiplexanalysis of one or more analytes in the same sample, or in two or moredifferent samples.

VI. Illustrative Labeling Reagents

It is to be understood that the below illustrated labeling reagents Aand B each represent one of many possible labeling reagents that can beprepared and utilized. It is also to be understood that the ordinarypractitioner, using no more that routine experimentation and thedisclosure provided herein, could easily produce other labeling reagentsof similar chemical structure. Accordingly, the disclosure is intendedto be illustrative and is not intended to either be exhaustive or to belimiting in any way.

Exemplary isotopically encoded isobaric sets A and B of compounds thatcan be produced are shown. It is to be understood that the belowillustrated labeling reagent sets each represent one of many possiblesets of labeling reagents that can be prepared and utilized. It is alsoto be understood that the ordinary practitioner, using no more thatroutine experimentation and the disclosure provided herein, could easilyproduce other sets of labeling reagents of similar chemical structures.Accordingly, the disclosure is intended to be illustrative and is notintended to either be exhaustive or to be limiting in any way.

Exemplary Set A:

Exemplary Set B:

Exemplary isotopically encoded mass differential sets C and D ofcompounds that can be produced are shown. It is to be understood thatthe below illustrated labeling reagent sets each represent one of manypossible sets of labeling reagents that can be prepared and utilized. Itis also to be understood that the ordinary practitioner, using no morethat routine experimentation and the disclosure provided herein, couldeasily produce other sets of labeling reagents of similar chemicalstructures. Accordingly, the disclosure is intended to be illustrativeand is not intended to either be exhaustive or to be limiting in anyway.

Exemplary Set C:

Exemplary Set D:

Other exemplary isotopically encoded compounds that can be producedusing the illustrated methods are described in this specification andthe associated figures and claims.

While the present teachings are described in conjunction with variousembodiments, it is not intended that the present teachings be limited tosuch embodiments. On the contrary, the present teachings encompassvarious alternatives, modifications and equivalents, as will beappreciated by those of skill in the art.

We claim:
 1. A labeling reagent for use in mass spectrometry,represented by formula I;

including a salt form and/or hydrate form thereof; wherein, Y is a 5, 6or 7 membered heterocyclic ring that may be substituted or unsubstitutedand that may optionally be cleavably linked to a support, wherein theheterocyclic ring comprises at least one ring nitrogen atom that islinked through a covalent bond to the group J; J is a group representedby formula —CJ′₂-, wherein each J′ is, independently of the other,hydrogen, deuterium, fluorine, chlorine, bromine, iodine, —R₃, —OR₃,—SR₃, —R₃′OR₃ or —R₃′SR₃; K is a group represented by formula—(CK′₂)_(n)— or —((CK′₂)_(m)—X₂—(CK′₂)_(m))_(p), wherein n is 0 or aninteger from 2 to 10, each in is, independently of the other, an integerfrom 1 to 5, p is an integer from 1 to 4 and each K′ is, independentlyof the other, hydrogen, deuterium, fluorine, chlorine, bromine, iodine,—R₄, —OR₄, —SR₄, —R₄′OR₄ or —R₄′SR₄; either 1) R₁ is hydrogen, deuteriumor R₆ and R₂ is hydrogen, deuterium or R₇; or 2) R₁ and R₂ takentogether is a group represented by formula —(CR′₂)_(q)— or—((CR′₂)_(m)—X₂—(CR′₂)_(m))_(p)— that forms a ring that bridges the twonitrogen atoms, wherein q is an integer from 1 to 10, each in is,independently of the other, an integer from 1 to 5, p is an integer from1 to 4 and each R′ is, independently of the other, hydrogen, deuterium,fluorine, chlorine, bromine, iodine, —R₅, —OR₅, —SR₅, —R₅′OR₅ or—R₅′SR₅; X₁ is ═O, ═S, ═NH or ═NR₇; each X₂ is, independently of theother, —O— or —S—; and Z is hydrogen or a covalently linked analyte;wherein, each R₃, R₄, R₅, R₆, and/or R₇ is, independently of the other,alkyl, alkenyl, alkynyl, aryl, heteroaryl or arylalkyl; each R₃′, R₄′,and/or R₅′ is, independently of the other, alkylene, alkenylene,alkynylene, arylene or alkylarylene; and the labeling reagent comprisesat least one isotopically enriched site.
 2. The labeling reagent ofclaim 1, wherein the group Y-J comprises at least one isotopicallyenriched site.
 3. The labeling reagent of claim 1, wherein the grouprepresented by formula I^(#);

comprises at least one isotopically enriched site; wherein, K is a grouprepresented by formula —(CK′₂)_(n)— or —((CK′₂)_(m)—X₂—(CK′₂)_(m))_(p),wherein n is 0 or an integer from 2 to 10, each m is, independently ofthe other, an integer from 1 to 5, p is an integer from 1 to 4 and eachK′ is, independently of the other, hydrogen, deuterium, fluorine,chlorine, bromine, iodine, —R₄, —OR₄, —SR₄, —R₄′OR₄ or —R₄′SR₄;either 1) R₁ is hydrogen, deuterium or R₆ and R₂ is hydrogen, deuteriumor R₇; or 2) R₁ and R₂ taken together is a group represented by formula—(CR′₂)_(q)— or —((CR′₂)_(m)—X²—(CR′₂)_(m))_(p)— that forms a ring thatbridges the two nitrogen atoms, wherein q is an integer from 1 to 10,each m is, independently of the other, an integer from 1 to 5, p is aninteger from 1 to 4 and each R′ is, independently of the other,hydrogen, deuterium, fluorine, chlorine, bromine, iodine, —R₅, —OR₅,—SR₅, —R₅′OR₅ or —R₅′SR₅; X₁ is ═O, ═S, ═NH or ═NR₇; and each X₂ is,independently of the other, —O— or —S—; wherein, each R₄, R₅, R₆, and/orR₇ is, independently of the other, alkyl, alkenyl, alkynyl, aryl,heteroaryl or arylalkyl; and each R₄′, and/or R₅′ is, independently ofthe other, alkylene, alkenylene, alkynylene, arylene or alkylarylene. 4.The labeling reagent of claim 1, wherein the group represented byformula Y-J comprises at least one isotopically enriched site and thegroup represented by formula I^(#);

comprises at least one isotopically enriched site; wherein, Y is a 5, 6or 7 membered heterocyclic ring that may be substituted or unsubstitutedand that may optionally be cleavably linked to a support, wherein theheterocyclic ring comprises at least one ring nitrogen atom that islinked through a covalent bond to the group J; J is a group representedby formula —CJ′₂-, wherein each J′ is, independently of the other,hydrogen, deuterium, fluorine, chlorine, bromine, iodine, —R₃, —OR₃,—SR₃, —R₃′OR₃ or —R₃′SR₃; K is a group represented by formula—(CK′₂)_(n)— or —((CK′₂)_(m)—X₂—(CK′₂)_(m))_(p), wherein n is 0 or aninteger from 2 to 10, each m is, independently of the other, an integerfrom 1 to 5, p is an integer from 1 to 4 and each K′ is, independentlyof the other, hydrogen, deuterium, fluorine, chlorine, bromine, iodine,—R₄, —OR₄, —SR₄, —R₄′OR₄ or —R₄′SR₄; either 1) R₁ is hydrogen, deuteriumor R₆, and R₂ is hydrogen, deuterium or R₇; or 2) R₁ and R₂ takentogether is a group represented by formula —(CR′₂)_(q)— or—((CR′₂)_(m)—X₂—(CR′₂)_(m))_(p)— that forms a ring that bridges the twonitrogen atoms, wherein q is an integer from 1 to 10, each in is,independently of the other, an integer from 1 to 5, p is an integer from1 to 4 and each R′ is, independently of the other, hydrogen, deuterium,fluorine, chlorine, bromine, iodine, —R₅, —OR₅, —SR₅, —R₅′OR₅ or—R₅′SR₅; X₁ is ═O, ═S, ═NH or ═NR₇; and each X₂ is, independently of theother, —O— or —S—; wherein, each R₃, R₄, R₅, R₆, and/or R₇ is,independently of the other, alkyl, alkenyl, alkynyl, aryl, heteroaryl orarylalkyl; and each R₃′, R₄′, and/or R₅′ is, independently of the other,alkylene, alkenylene, alkynylene, arylene or alkylarylene.
 5. Thelabeling reagent of claim 1, represented by formula III*;

including a salt form and/or hydrate form thereof; wherein, R₁₁ ishydrogen, deuterium, methyl, —C(H)₂D, —C(H)D₂, —CD₃ or —R′″, Z ishydrogen or a covalently linked analyte; wherein, R′″ is H₂N—R₉′—,H(R₁₀)N—R₉′—, (R₁₀)₂N—R₉′—, HO—R₉′—, HS—R₉′— or a cleavable linker thatcleavably links the compound to a support; and wherein, each R₉′ is,independently of the other, alkylene, alkenylene, alkynylene, arylene oralkylarylene; and each R₁₀ is, independently of the other, alkyl,alkenyl, alkynyl, aryl, heteroaryl or arylalkyl.
 6. The labeling reagentof claim 5, wherein the labeling reagent is, represented by formula M,MI, MII, MIII, MIV or MV;

including a salt form and/or hydrate form thereof; wherein, * indicatesan isotopically enriched site comprising a ¹³C substituted for ¹²C, ¹⁵Nsubstituted for ¹⁴N or ¹⁸O substituted for ¹⁶O, as appropriate; and Z ishydrogen or a covalently linked analyte.
 7. The labeling reagent ofclaim 5, wherein the labeling reagent is represented by formula MVI,MVII, MVIII, MIX, MX, MXI, MXII or MXIII:

including a salt form and/or hydrate form thereof; wherein, * indicatesan isotopically enriched site comprising a ¹³C substituted for ¹²C or¹⁵N substituted for ¹⁴N, as appropriate; and Z is hydrogen or acovalently linked analyte.
 8. A labeling reagent for use in massspectrometry represented by formula B:

wherein the labeling reagent comprises at least one isotopicallyenriched site.
 9. A set of isobaric labeling reagents, comprising thereagents represented by formula BI, BII, BIII and BIV:

wherein * indicates an isotopically enriched site comprising a ¹³Csubstituted for ¹²C, ¹⁵N substituted for ¹⁴N or ¹⁸O substituted for ¹⁶O,as appropriate.
 10. A set of structurally similar labeling reagents ofdifferent gross mass, comprising the reagents represented by formula BIand BV:

wherein * indicates an isotopically enriched site comprising a ¹³Csubstituted for ¹²C or ¹⁵N substituted, as appropriate.
 11. The labelingreagent of claim 1, wherein Z is a covalently linked analyte and areactive group of the analyte is a carboxylic acid.
 12. The labelingreagent of claim 11, wherein said covalently linked analyte is selectedfrom the group consisting of prostaglandins, fatty acids, carnitines,and salts thereof.